Eyeblink Conditioning In Rats Research Articles

Impact of fornix lesions on tone-off delay vs tone-on trace eyeblink conditioning in rats

Research has shown differences in the neural mechanisms that support trace and delay eyeblink conditioning. The present experiment furthered this investigation by examining the effect of electrolytic fornix lesions on acquisition of trace and delay eyeblink conditioning in the rat. Importantly, the conditioned stimulus (CS) for trace conditioning was a standard tone-on cue, and the CS for delay conditioning was either a tone-off or tone-on CS. The results showed that fornix lesions impaired trace-, but not delay conditioning in rats trained with the tone-on CS or tone-off CS. The findings are consistent with previous studies that found trace-, but not delay eyeblink conditioning is a hippocampal dependent form of associative learning. Our results also indicate that the neural pathways for tone-off delay conditioning and tone-on trace conditioning are different, even though the structural composition of a tone-off CS and the trace conditioning interval are the same cue (i.e., the absence of sound). These findings indicate that the absence of a sensory cue (i.e., tone-off CS) and the presence of a sensory cue (i.e., tone-on CS) have equivalent associative value and effectiveness for engaging the neural pathways that support delay eyeblink conditioning.

Intracerebellar infusion of an mGluR1/5 agonist enhances eyeblink conditioning.

Cerebellar metabotropic glutamate receptor 1 (mGluR1) expressed by Purkinje cells may play an important role in learning-related cerebellar plasticity. Eyeblink conditioning (EBC) is a well-studied form of Pavlovian learning that engages discrete areas of cerebellar cortex and deep cerebellar nuclei. EBC is impaired in mGluR1 knockout mice. Here, we show that infusion of the mGluR1/5 agonist DHPG into the lobulus simplex region of cerebellar cortex facilitates EBC in rats. Infusion was made prior to Sessions 1 and 2 of EBC but the facilitatory effects persisted throughout subsequent, noninfusion sessions. The facilitatory effects were confined to frequency of eyeblink conditioned responses (CRs); there were no effects on amplitude or latency of CRs. There were also no effects on reflexive responding to the tone conditioned stimulus or eyelid stimulation unconditioned stimulus. The current results provide further evidence that cerebellar mGluR1 plays a role in cerebellar-dependent associative learning and complement previous studies using mGluR1 knockout mice. (PsycInfo Database Record (c) 2021 APA, all rights reserved).

Sensory system development influences the ontogeny of hippocampal associative coding and trace eyeblink conditioning

Until recently, it was believed that hippocampal development was the primary rate-limiting factor in the developmental emergence of hippocampal forms of learning, such as trace eyeblink conditioning (EBC). Indeed, hippocampal neuronal activity shows an age-related increase in both complexity and task responsiveness during trace EBC. However, recent work from our laboratory suggests that sensory system development may also play a role. Training with the earlier-developing somatosensory system results in an earlier emergence of trace EBC in rats, suggesting that the development of sensory input to the hippocampus may influence the development of trace EBC. The goal of the current study was to examine the activity of hippocampal CA1 pyramidal cells during acquisition of trace EBC with an early-developing somatosensory CS. Rat pups were trained with a vibration CS on postnatal days (P) 17-19, P21-23, and P24-26 while CA1 pyramidal cell activity was recorded. Results indicated that CA1 neurons show an age-related increase in responsiveness to trial events. Although the magnitude of neuronal responding showed age-related increases in activity, all three age groups demonstrated learning-related increases in firing rate magnitude and peaks in firing rate were evident both at CS onset and offset. These findings suggest that the ontogeny of trace eyeblink conditioning is related to both hippocampal and sensory system development.

Cannabinoid modulation of memory consolidation within the cerebellum

Cannabinoid receptors contribute to learning and synaptic plasticity mechanisms. The cerebellum contains a high density of cannabinoid receptors and manipulations of cannabinoid receptors affect synaptic plasticity within the cerebellar cortex. In vivo studies have found that cannabinoid agonists impair learning of cerebellum-dependent eyeblink conditioning in rodents and humans. However, the role of cannabinoid receptors or endocannabinoids in memory consolidation within the cerebellum has not been examined. In the current study, we examined the role of cannabinoid receptors and endocannabinoids during learning and consolidation of eyeblink conditioning in rats. Administration of the cannabinoid receptor agonist WIN55,212-2 or drugs that increase/decrease endocannabinoid levels directly into the cerebellar cortex before each training session resulted in marked learning impairments. When administered 1 h after each training session, during memory consolidation, the cannabinoid inverse agonist SR141716A or the endocannabinoid suppressor THL impaired memory. In contrast, increasing endocannabinoid levels with JZL-184 or infusion of WIN55,212-2 within the cerebellar cortex facilitated memory consolidation 1h post-training. Intracerebellar manipulations of cannabinoid receptors or endocannabinoid levels had no effect on memory consolidation when administered 3 or 6h after each training session. The results demonstrate that cannabinoids impair cerebellar learning, but facilitate memory consolidation mechanisms within the cerebellar cortex 1–3h after training.

Electrophysiological signatures of learning in the hippocampus

Electrophysiological signatures of learning in the hippocampus

Cross-modal savings in the contralateral eyelid conditioned response.

The present experiment monitored bilateral eyelid responses during eyeblink conditioning in rats trained with a unilateral unconditioned stimulus (US). Three groups of rats were used to determine if cross-modal savings occurs when the location of the US is switched from one eye to the other. Rats in each group first received paired or unpaired eyeblink conditioning with a conditioned stimulus (tone or light; conditional stimulus [CS]) and a unilateral periorbital electrical stimulation US. All rats were subsequently given paired training, but with the US location (Group 1), CS modality (Group 2), or US location and CS modality (Group 3) changed. Changing the location of the US alone resulted in an immediate transfer of responding in both eyelids (Group 1) in rats that received paired training before the transfer session. Rats in Groups 2 and 3 that initially received paired training showed facilitated learning to the new CS modality during the transfer sessions, indicating that cross-modal savings occurs whether or not the location of the US is changed. All rats that were initially given unpaired training acquired conditioned eyeblink responses similar to de novo acquisition rate during the transfer sessions. Savings of CR incidence was more robust than savings of CR amplitude when the US switched sides, a finding that has implications for elucidating the neural mechanisms of cross-modal savings.

Localization of the cerebellar cortical zone mediating acquisition of eyeblink conditioning in rats

Delay eyeblink conditioning is established by paired presentations of a conditioned stimulus (CS) such as a tone or light and an unconditioned stimulus (US) that elicits eyelid closure before training. The CS and US inputs converge on Purkinje cells in the cerebellar cortex. The cerebellar cortex plays a substantial role in acquisition of delay eyeblink conditioning in rabbits and rodents, but the specific area of the cortex that is necessary for acquisition in rodents has not been identified. A recent study identified an eyeblink microzone in the mouse cerebellar cortex at the base of the primary fissure (Heiney, Kim, Augustine, & Medina, 2014). There is no evidence that the cortex in this eyeblink microzone plays a role in rodent eyeblink conditioning but it is a good candidate region. Experiment 1 examined the effects of unilateral (ipsilateral to the US) lesions of lobule HVI, the lateral anterior lobe, or the base of the primary fissure on eyeblink conditioning in rats. Lesions of either the anterior lobe or lobule HVI impaired acquisition, but lesions of the base of the primary fissure produced the largest deficit. Experiment 2 used reversible inactivation with muscimol to demonstrate that inactivation of the putative eyeblink microzone severely impaired acquisition and had only a modest effect on retention of eyeblink conditioning. The findings indicate that the base of the primary fissure is the critical zone of the cerebellar cortex for acquisition of eyeblink conditioning in rats.

Cellular Mechanisms and Behavioral Consequences of Kv1.2 Regulation in the Rat Cerebellum

The potassium channel Kv1.2 α-subunit is expressed in cerebellar Purkinje cell (PC) dendrites where its pharmacological inhibition increases excitability (Khavandgar et al., 2005). Kv1.2 is also expressed in cerebellar basket cell (BC) axon terminals (Sheng et al., 1994), where its blockade increases BC inhibition of PCs (Southan and Robertson, 1998a). Secretin receptors are also expressed both in PC dendrites and BC axon terminals (for review, see (Yuan et al., 2011). The effect of secretin on PC excitability is not yet known, but, like Kv1.2 inhibitors, secretin potently increases inhibitory input to PCs (Yung et al., 2001). This suggests secretin may act in part by suppressing Kv1.2. Receptor-mediated endocytosis is a mechanism of Kv1.2 suppression (Nesti et al., 2004). This process can be regulated by protein kinase A (PKA) (Connors et al., 2008). Since secretin receptors activate PKA (Wessels-Reiker et al., 1993), we tested the hypothesis that secretin regulates Kv1.2 trafficking in the cerebellum. Using cell-surface protein biotinylation of rat cerebellar slices, we found secretin decreased cell-surface Kv1.2 levels by modulating Kv1.2 endocytic trafficking. This effect was mimicked by activating adenylate cyclase (AC) with forskolin, and was blocked by pharmacological inhibitors of AC or PKA. Imaging studies identified the BC axon terminal and PC dendrites as loci of AC-dependent Kv1.2 trafficking. The physiological significance of secretin-regulated Kv1.2 endocytosis is supported by our finding that infusion into the cerebellar cortex of either the Kv1.2 inhibitor tityustoxin-Kα, or of the Kv1.2 regulator secretin, significantly enhances acquisition of eyeblink conditioning in rats.

Increasing SK2 channel activity impairs associative learning

Enhanced intrinsic neuronal excitability of hippocampal pyramidal neurons via reductions in the postburst afterhyperpolarization (AHP) has been hypothesized to be a biomarker of successful learning. This is supported by considerable evidence that pharmacologic enhancement of neuronal excitability facilitates learning. However, it has yet to be demonstrated that pharmacologic reduction of neuronal excitability restricted to the hippocampus can retard acquisition of a hippocampus-dependent task. Thus, the present study was designed to address this latter point using a small conductance potassium (SK) channel activator NS309 focally applied to the dorsal hippocampus. SK channels are important contributors to intrinsic excitability, as measured by the medium postburst AHP. NS309 increased the medium AHP and reduced excitatory postsynaptic potential width of CA1 neurons in vitro. In vivo, NS309 reduced the spontaneous firing rate of CA1 pyramidal neurons and impaired trace eyeblink conditioning in rats. Conversely, trace eyeblink conditioning reduced levels of SK2 channel mRNA and protein in the hippocampus. Therefore, the present findings indicate that modulation of SK channels is an important cellular mechanism for associative learning and further support postburst AHP reductions in hippocampal pyramidal neurons as a biomarker of successful learning.

Retention and extinction of delay eyeblink conditioning are modulated by central cannabinoids

Rats administered the cannabinoid agonist WIN55,212-2 or the antagonist SR141716A exhibit marked deficits during acquisition of delay eyeblink conditioning, as noted by Steinmetz and Freeman in an earlier study. However, the effects of these drugs on retention and extinction of eyeblink conditioning have not been assessed. The present study examined the effects of WIN55,212-2 and SR141716A on retention and extinction of delay eyeblink conditioning in rats. Rats were given acquisition training for five daily sessions followed by one session of retention training with subcutaneous administration of 3 mg/kg of WIN55,212-2 or 5 mg/kg of SR141716A and an additional session with the vehicle. Two sessions of extinction training were then given with WIN55,212-2, SR141716A, or vehicle. Retention and extinction were impaired by WIN55,212-2, whereas SR141716A produced no deficits. The extinction deficit in rats given WIN55,212-2 was observed only during the first session, suggesting a specific impairment in short-term plasticity mechanisms. The current results and previous findings indicate that the cannabinoid system modulates cerebellar contributions to acquisition, retention, and extinction of eyeblink conditioning.

Neuronal Correlates of Cross-Modal Transfer in the Cerebellum and Pontine Nuclei

Cross-modal transfer occurs when learning established with a stimulus from one sensory modality facilitates subsequent learning with a new stimulus from a different sensory modality. The current study examined neuronal correlates of cross-modal transfer of pavlovian eyeblink conditioning in rats. Neuronal activity was recorded from tetrodes within the anterior interpositus nucleus (IPN) of the cerebellum and basilar pontine nucleus (PN) during different phases of training. After stimulus preexposure and unpaired training sessions with a tone conditioned stimulus (CS), light CS, and periorbital stimulation unconditioned stimulus (US), rats received associative training with one of the CSs and the US (CS1-US). Training then continued on the same day with the other CS to assess cross-modal transfer (CS2-US). The final training session included associative training with both CSs on separate trials to establish stronger cross-modal transfer (CS1/CS2). Neurons in the IPN and PN showed primarily unimodal responses during pretraining sessions. Learning-related facilitation of activity correlated with the conditioned response (CR) developed in the IPN and PN during CS1-US training. Subsequent CS2-US training resulted in acquisition of CRs and learning-related neuronal activity in the IPN but substantially less little learning-related activity in the PN. Additional CS1/CS2 training increased CRs and learning-related activity in the IPN and PN during CS2-US trials. The findings suggest that cross-modal neuronal plasticity in the PN is driven by excitatory feedback from the IPN to the PN. Interacting plasticity mechanisms in the IPN and PN may underlie behavioral cross-modal transfer in eyeblink conditioning.

The effects of two forms of physical activity on eyeblink classical conditioning

Voluntary exercise, in the form of free access to a running wheel in the home cage, has been shown to improve several forms of learning and memory. Acrobatic training, in the form of learning to traverse an elevated obstacle course, has been shown to induce markers of neural plasticity in the cerebellar cortex in rodents. In three experiments, we examined the effects of these two forms of physical activity on delay eyeblink conditioning in rats. In Experiment 1, exercising rats were given 17 days of free access to a running wheel in their home cage prior to 10 days of delay eyeblink conditioning. Rats that exercised conditioned significantly better and showed a larger reflexive eyeblink unconditioned response to the periocular stimulation unconditioned stimulus than rats that did not exercise. In Experiment 2, exercising rats were given 17 days of free access to a running wheel in their home cage prior to 10 days of explicitly unpaired stimulus presentations. Rats that exercised responded the same to tone, light, and periocular stimulation as rats that did not exercise. In Experiment 3, acrobatic training rats were given 15 days of daily training on an elevated obstacle course prior to 10 days of eyeblink conditioning. Activity control rats underwent 15 days of yoked daily running in an open field. Rats that underwent acrobatic training did not differ in eyeblink conditioning from activity control rats. The ability to measure the learned response precisely, and the well-mapped neural circuitry of eyeblink conditioning offer some advantages for the study of exercise effects on learning and memory.

Central cannabinoid receptors modulate acquisition of eyeblink conditioning

Delay eyeblink conditioning is established by paired presentations of a conditioned stimulus (CS) such as a tone or light, and an unconditioned stimulus (US) that elicits the blink reflex. Conditioned stimulus information is projected from the basilar pontine nuclei to the cerebellar interpositus nucleus and cortex. The cerebellar cortex, particularly the molecular layer, contains a high density of cannabinoid receptors (CB1R). The CB1Rs are located on the axon terminals of parallel fibers, stellate cells, and basket cells where they inhibit neurotransmitter release. The present study examined the effects of a CB1R agonist WIN55,212-2 and antagonist SR141716A on the acquisition of delay eyeblink conditioning in rats. Rats were given subcutaneous administration of 1, 2, or 3 mg/kg of WIN55,212-2 or 1, 3, or 5 mg/kg of SR141716A before each day of acquisition training (10 sessions). Dose-dependent impairments in acquisition were found for WIN55,212-2 and SR141716A, with no effects on spontaneous or nonassociative blinking. However, the magnitude of impairment was greater for WIN55,212-2 than SR141716A. Dose-dependent impairments in conditioned blink response (CR) amplitude and timing were found with WIN55,212-2 but not with SR141716A. The findings support the hypothesis that CB1Rs in the cerebellar cortex play an important role in plasticity mechanisms underlying eyeblink conditioning.

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.

Ventral lateral geniculate input to the medial pons is necessary for visual eyeblink conditioning in rats.

The conditioned stimulus (CS) pathway that is necessary for visual delay eyeblink conditioning was investigated in the current study. Rats were initially given eyeblink conditioning with stimulation of the ventral nucleus of the lateral geniculate (LGNv) as the CS followed by conditioning with light and tone CSs in separate training phases. Muscimol was infused into the medial pontine nuclei (MPN) after each training phase to examine conditioned response (CR) retention to each CS. The spread of muscimol infusions targeting the MPN was examined with fluorescent muscimol. Muscimol infusions into the MPN resulted in a severe impairment in retention of CRs with the LGNv stimulation and light CSs. A less severe impairment was observed with the tone CS. The results suggest that CS information from the LGNv and light CSs is relayed to the cerebellum through the MPN. Retrograde tracing with fluoro-gold (FG) showed that the LGNv and nucleus of the optic tract have ipsilateral projections to the MPN. Unilateral inputs to the MPN from the LGNv and nucleus of the optic tract may be part of the visual CS pathway that is necessary for visual eyeblink conditioning.

Stimulation of the lateral geniculate, superior colliculus, or visual cortex is sufficient for eyeblink conditioning in rats.

The role of the cerebellum in eyeblink conditioning is well established. Less work has been done to identify the necessary conditioned stimulus (CS) pathways that project sensory information to the cerebellum. A possible visual CS pathway has been hypothesized that consists of parallel inputs to the pontine nuclei from the lateral geniculate nucleus (LGN), superior colliculus (SC), pretectal nuclei, and visual cortex (VCTX) as reported by Koutalidis and colleagues in an earlier paper. The following experiments examined whether electrical stimulation of neural structures in the putative visual CS pathway can serve as a sufficient CS for eyeblink conditioning in rats. Unilateral stimulation of the ventral LGN (Experiment 1), SC (Experiment 2), or VCTX (Experiment 3) was used as a CS paired with a periorbital shock unconditioned stimulus. Stimulation was delivered to the hemisphere contralateral to the conditioned eye. Rats in all experiments were given five 100-trial sessions of paired or unpaired eyeblink conditioning with the stimulation CS followed by three paired sessions with a light CS. Stimulation of each visual area when paired with the unconditioned stimulus supported acquisition of eyeblink conditioned responses (CRs) and substantial savings when switched to a light CS. The results provide evidence for a unilateral parallel visual CS pathway for eyeblink conditioning that includes the LGN, SC, and VCTX inputs to the pontine nuclei.

Examination of bilateral eyeblink conditioning in rats.

This experiment monitored eyelid responses bilaterally during delay eyeblink conditioning in rats. Rats were given paired or unpaired training with a tone or light conditioned stimulus (CS) and a unilateral periorbital shock unconditioned stimulus (US). Rats given paired training acquired high levels of conditioned responses (CRs), which occurred in both eyelids. However, acquisition was faster, and the overall percentage of CRs was greater in the eyelid that was ipsilateral to the US. CRs in the eyelid ipsilateral to the US also had shorter onset latencies and larger amplitudes than CRs in the contralateral eyelid. Both eyelids consistently showed high percentages of unconditioned responses (UR) to the US, and the UR amplitude decreased across training sessions in the paired group. The present study demonstrated that CRs occur robustly in both eyelids of rats given eyeblink conditioning, which is similar to previous findings in humans and monkeys. The results also showed that conditioning occurs more prominently in the eyelid that is ipsilateral to the US, which is similar to previous findings in humans, monkeys, dogs, and rabbits.

Ontogenetic change in the auditory conditioned stimulus pathway for eyeblink conditioning

Two experiments examined the neural mechanisms underlying the ontogenetic emergence of auditory eyeblink conditioning. Previous studies found that the medial auditory thalamus is necessary for eyeblink conditioning with an auditory conditioned stimulus (CS) in adult rats. In experiment 1, stimulation of the medial auditory thalamus was used as a CS in rat pups trained on postnatal days (P) 17-18, 24-25, or 31-32. All three age groups showed significant acquisition relative to unpaired controls. However, there was an age-related increase in the rate of conditioning. Experiment 2 examined the effect of inactivating the medial auditory thalamus with muscimol on auditory eyeblink conditioning in rats trained on P17-18, 24-25, or 31-32. Rat pups trained on P24-25 and P31-32, but not P17-18, showed a significant reduction in conditioned responses following muscimol infusions. The findings suggest that the thalamic contribution to auditory eyeblink conditioning continues to develop through the first postnatal month.

Eyeblink conditioning using cochlear nucleus stimulation as a conditioned stimulus in developing rats

Previous studies demonstrated that the development of auditory conditioned stimulus (CS) input to the cerebellum may be a neural mechanism underlying the ontogenetic emergence of eyeblink conditioning in rats. The current study investigated the role of developmental changes in the projections of the cochlear nucleus (CN) in the ontogeny of eyeblink conditioning using electrical stimulation of the CN as a CS. Rat pups were implanted with a bipolar stimulating electrode in the CN and given six 100-trial training sessions with a 300 ms stimulation train in the CN paired with a 10 ms periorbital shock unconditioned stimulus (US) on postnatal days (P) 17-18 or 24-25. Control groups were given unpaired presentations of the CS and US. Rats in both age groups that received paired training showed significant increases in eyeblink conditioned responses across training relative to the unpaired groups. The rats trained on P24-25, however, showed stronger conditioning relative to the group trained on P17-18. Rats with missed electrodes in the inferior cerebellar peduncle or in the cerebellar cortex did not show conditioning. The findings suggest that developmental changes in the CN projections to the pons, inferior colliculus, or medial auditory thalamus may be a neural mechanism underlying the ontogeny of auditory eyeblink conditioning.

Inferior colliculus lesions impair eyeblink conditioning in rats.

The neural plasticity necessary for acquisition and retention of eyeblink conditioning has been localized to the cerebellum. However, the sources of sensory input to the cerebellum that are necessary for establishing learning-related plasticity have not been identified completely. The inferior colliculus may be a source of sensory input to the cerebellum through its projection to the medial auditory thalamus. The medial auditory thalamus is necessary for eyeblink conditioning in rats and projects to the lateral pontine nuclei, which then project to the cerebellar nuclei and cortex. The current experiment examined the role of the inferior colliculus in auditory eyeblink conditioning. Rats were given bilateral or unilateral (contralateral to the conditioned eye) lesions of the inferior colliculus prior to 10 d of delay eyeblink conditioning with a tone CS. Rats with bilateral or unilateral lesions showed equivalently impaired acquisition. The extent of damage to the contralateral inferior colliculus correlated with several measures of conditioning. The findings indicate that the contralateral inferior colliculus provides auditory input to the cerebellum that is necessary for eyeblink conditioning.