Differential effects of acute stress on spatial learning and memory in the open-field tower maze across the female estrous cycle.

The purpose of the present investigation was to test how acute stress and levels of circulating estrogens together influence acquisition and retention of spatial learning, as well as explorative behaviors in female rats. We used the hippocampus-dependent Open-field Tower Maze (OFTM) task to assess acquisition followed by a retention test (reacquisition) that was given 48 h later. Immediately prior to acquisition, experimental rats were exposed to an acute restraint stress and were trained under bright lights. Female rats' estrous cycles were tracked throughout training and testing. Exposure to stress did not affect learning when levels of estrogens were low (i.e., during estrus and metestrus). However, acute stress exposure significantly lowered spatial acquisition of the female rats in the phases with rising levels of estrogens (i.e., during diestrus and proestrus). Furthermore, this stress-induced diminishment during acquisition was evident at the beginning of the retention without any presentation of stress. The present findings provide insight about the interactive relationship between stress and sex hormones on cognitive functions.

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.

Fornix lesions impair place-, but not response-learning in the open-field tower maze.

The purpose of the present study was to examine hippocampal function for spatial learning in a land-based circular maze (i.e., the open-field tower maze [OFTM]). The OFTM, a task designed to be non-stressful, has been previously used to demonstrate the influence of gonadal hormones on spatial learning. Thus, determination of brain function for navigating in the OFTM provides an important extension to previous knowledge. Fornix lesions were used in the present experiment to disrupt hippocampal processing. After initial pre-training, rats received either an electrolytic fornix lesion surgery or a sham surgery. The rats from each surgical group were given either place- or response-training in the OFTM. The results showed that (1) lesioned place-learners required more trials than sham place-learners to solve the OFTM and (2) lesioned response-learners solved the OFTM at the same rate as sham response-learners. Our findings support the hypothesis that the hippocampus is necessary for place-, but not response-learning in the OFTM task. The OFTM is an appetitive task that does not depend on a choice between restricted directions that a rat would be required to make in a T-maze or a radial arm-maze, and does not include aversive components inherent to a Morris Water Maze or Barnes Maze. Thus, the OFTM can be used to investigate the manipulations of hippocampus-dependent spatial learning without confounding variables related to an animal's stress level.

Sex differences and the role of acute stress in the open-field tower maze.

Many studies provide evidence that differences in spatial learning exist between males and females. However, it is necessary to consider non-mnemonic factors that may influence these findings. The present experiment investigated acquisition, retention, and the effects of stress on response- and place-learning in male and female rats. Rats were trained in an open-field tower maze. Procedures were used to minimize stress in the rats, and their ability to solve place- or response-learning in the maze was determined by analyzing a response variable (i.e., first choice correct response) that was not influenced by general locomotor activity. The results revealed that male and female rats acquire place- and response-learning at the same rate even though females moved significantly faster in the maze. However, females showed better retrieval of place-, but not response-learning compared to male rats. This effect appeared to be enhanced when the rats were tested immediately following an acute restraint stress. Furthermore, both female and male rats that were exposed to acute restraint stress showed less impairment than controls when subsequently tested in a novel situation. These findings have clinical implications that a mild physiological stress response can make one more cognitively resistant to adversities later in life.

Auditory cue absence as a conditioned stimulus for delay eyeblink conditioning.

The present experiment was designed to determine if the absence of an auditory cue (i.e., a "tone-off" cue) would be an effective conditioned stimulus (CS) for delay eyeblink conditioning and to test if the medial geniculate nucleus (MGN) is part of the sensory pathway for tone-off conditioning. Rats were given paired or unpaired delay eyeblink conditioning to examine if responding to a tone-off CS was due to an associative process. An inactivation technique was performed on a separate group of rats to determine if the MGN is needed to express tone-off conditioning. The results showed that rats given paired conditioning acquired robust conditioned eyeblink responses (CRs) compared with rats given unpaired conditioning and that expression of tone-off elicited CRs was impaired when the MGN was inactivated. The findings suggest that tone-on and tone-off eyeblink conditioning may share a common neural pathway. (PsycINFO Database Record

The Miracle Fruit: An Undergraduate Laboratory Exercise in Taste Sensation and Perception.

"Miracle Fruit" is a taste-altering berry that causes sour foods to be perceived as sweet. The present paper describes a laboratory exercise that uses Miracle Fruit to educate students about the sensation and perception of taste. This laboratory exercise reinforces course material pertaining to the function of sweet taste receptors covered in a Sensation and Perception course at Christopher Newport University. Here we provide a step-by-step explanation of the methodology, and an example of data collected and analyzed by one group of students who participated in this laboratory exercise. The origins of the Miracle Fruit, the structure and the physiological function of miraculin (the glycoprotein responsible for the taste-modifying effect found in the pulp of the Miracle Fruit) were discussed before the laboratory exercise. Students then sampled foods known to target different types of tastes (i.e., sweet, sour, bitter and salty) and rated their perception of taste intensity for each food item. Next, students each consumed Miracle Fruit berries, then resampled each original food item and again recorded their perception of taste intensity ratings for these foods. The data confirmed that the sour food items were perceived sweeter after the Miracle Fruit was consumed. The students also completed a written assignment to assess what they learned about the origins, structure, and physiological function of Miracle Fruit. This hands-on laboratory exercise received positive feedback from students. The exercise can be used by other neuroscience educators to teach concepts related to the sensory system of taste.

Place and Response Learning in the Open-field Tower Maze.

This protocol describes how the Open-field Tower Maze (OFTM) paradigm is used to study spatial learning in rodents. This maze is especially useful for examining how rats learn to use a place- or response-learning to successfully navigate in an open-field arena. Additionally, this protocol describes how the OFTM differs from other behavioral maze paradigms that are commonly used to study spatial learning in rodents. The OFTM described in this article was adapted from the one previously described by Cole, Clipperton, and Walt (2007). Specifically, the OFTM was created to test spatial learning in rodents without the experimenter having to consider how "stress" might play a role as a confounding variable. Experiments have shown that stress-alone can significantly affect cognitive function(1). The representative results section contains data from an experiment that used the OFTM to examine the effects of estradiol treatment on place- and response-learning in adult female Sprague Dawley rats(2). Future studies will be designed to examine the role of the hippocampus and striatum in place- and response-learning in the OFTM.

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.

Learning-related neuronal activity in the ventral lateral geniculate nucleus during associative cerebellar learning.

During delay eyeblink conditioning, rats learn to produce an eyelid-closure conditioned response (CR) to a conditioned stimulus (CS), such as a light, which precedes and coterminates with an unconditioned stimulus (US). Previous studies have suggested that the ventral lateral geniculate nucleus (LGNv) might play an important role in visual eyeblink conditioning by supplying visual sensory input to the pontine nuclei (PN) and also receiving feedback from the cerebellum. No prior study has investigated LGNv neuronal activity during eyeblink conditioning. The present study used multiple tetrodes to monitor single-unit activity in the rat LGNv during pre-exposure (CS only), unpaired CS/US, and paired CS-US training conditions. This behavioral-training sequence was used to investigate nonassociative- and associative-driven neuronal activity in the LGNv during training. LGNv neuronal activity habituated during unpaired training and then recovered from habituation during subsequent paired training, which may indicate that the LGNv plays a role in attention to the CS. The amplitude of LGNv neuronal activity correlated with CR production during paired but not unpaired CS/US training. Cerebellar feedback to the LGNv may play a role in modulating LGNv activity and attention to the CS during paired training. Based on the present findings, we hypothesize that the role of LGNv in visual eyeblink conditioning goes beyond simply routing visual CS information to the PN and involves modulation of attention.

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.

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.

Cerebellar inactivation impairs cross modal savings of eyeblink conditioning.

Eyeblink conditioning using a conditioned stimulus (CS) from one sensory modality (e.g., an auditory CS) is greatly enhanced when the subject is previously trained with a CS from a different sensory modality (e.g., a visual CS). The enhanced acquisition to the second modality CS results from cross modal savings. The current study was designed to examine the role of the cerebellum in establishing cross modal savings in eyeblink conditioning with rats. In the first experiment rats were given paired or unpaired presentations with a CS (tone or light) and an unconditioned stimulus. All rats were then given paired training with a different modality CS. Only rats given paired training showed cross modal savings to the second modality CS. Experiment 2 showed that cerebellar inactivation during initial acquisition to the first modality CS completely prevented savings when training was switched to the second modality CS. Experiment 3 showed that cerebellar inactivation during initial cross modal training also prevented savings to the second modality stimulus. These results indicate that the cerebellum plays an essential role in establishing cross modal savings of eyeblink conditioning.

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.

Changes in inhibition during differential eyeblink conditioning with increased training.

Three experiments examined inhibitory learning in rats, using Pavlovian and differential inhibitory eyeblink conditioning procedures. Experiment 1 was designed to compare summation and retardation effects following Pavlovian conditioned inhibition (A+/XA-) or differential inhibition (A+/X-) training using auditory and visual conditioned stimuli (CSs). After ten 100-trial sessions of training, both Pavlovian conditioned inhibition and differential inhibition produced a retardation effect. However, a summation effect was obtained only for rats given Pavlovian conditioned inhibition training. Experiment 2 showed that increasing differential inhibition training to twenty 100-trial sessions produced summation and retardation effects. In Experiment 3, rats were trained with either ten or twenty 100-trial sessions of intramodal inhibitory training with two tone CSs (2 kHz vs. 8 kHz). Summation and retardation effects were obtained after only 20 sessions of differential conditioning. The findings indicate that extensive training is needed to establish conditioned inhibition with intermodal or intramodal differential conditioning.

Eyeblink conditioning in 12-day-old rats using pontine stimulation as the conditioned stimulus.

A fundamental issue in developmental science is whether ontogenetic changes in memory are caused by the development of cellular plasticity mechanisms within the brain's memory systems or maturation of sensory inputs to the memory systems. Here, we provide evidence that the development of eyeblink conditioning, a form of associative learning that depends on the cerebellum, is driven by the development of sensory inputs rather than the development of neuronal plasticity mechanisms. We find that rats as young as 12 days old show associative eyeblink conditioning when pontine stimulation is used in place of an external (e.g., a tone) conditioned stimulus. Eyeblink-conditioned responses established with pontine stimulation in 12-day-old rats were reversibly abolished by an infusion of muscimol into the cerebellar interpositus nucleus. The findings suggest that cerebellar neurons are capable of supporting associative learning-specific plasticity in vivo in very immature animals if given sufficient afferent stimulation.

Medial auditory thalamic stimulation as a conditioned stimulus for eyeblink conditioning in rats.

The neural pathways that convey conditioned stimulus (CS) information to the cerebellum during eyeblink conditioning have not been fully delineated. It is well established that pontine mossy fiber inputs to the cerebellum convey CS-related stimulation for different sensory modalities (e.g., auditory, visual, tactile). Less is known about the sources of sensory input to the pons that are important for eyeblink conditioning. The first experiment of the current study was designed to determine whether electrical stimulation of the medial auditory thalamic nuclei is a sufficient CS for establishing eyeblink conditioning in rats. The second experiment used anterograde and retrograde tract tracing techniques to assess neuroanatomical connections between the medial auditory thalamus and pontine nuclei. Stimulation of the medial auditory thalamus was a very effective CS for eyeblink conditioning in rats, and the medial auditory thalamus has direct ipsilateral projections to the pontine nuclei. The results suggest that the medial auditory thalamic nuclei and their projections to the pontine nuclei are components of the auditory CS pathway in eyeblink conditioning.

Perirhinal cortex lesions impair simultaneous but not serial feature-positive discrimination learning.

The role of the perirhinal cortex in discriminative eyeblink conditioning was examined by means of feature-positive discrimination procedures with simultaneous (A-/XA+) and serial (A-/X-->A+) stimulus compounds. Lesions of the perirhinal cortex severely impaired acquisition of simultaneous feature-positive discrimination but produced no impairment in serial feature-positive discrimination. The results suggest that the perirhinal cortex plays a role in discriminative eyeblink conditioning by resolving ambiguity in discriminations with overlapping stimulus elements.

Perirhinal cortex lesions impair feature-negative discrimination.

The role of the perirhinal cortex in inhibitory eyeblink conditioning was examined. In Experiment 1, rats were given lesions of the perirhinal cortex or control surgery and subsequently trained with a feature-negative discrimination procedure followed by summation and retardation tests for conditioned inhibition. Perirhinal cortex lesions impaired, but did not prevent acquisition of feature-negative discrimination. Results from the summation test showed that rats with perirhinal cortex lesions could not generalize feature-negative discrimination to a new stimulus. There were no group differences during the retardation test. Experiment 2 showed that lesions of the perirhinal cortex did not impair simple excitatory conditioning. Experiment 3 showed that perirhinal cortex lesions had no effect on acquisition of a simple tone-light discrimination. The results suggest that the perirhinal cortex plays a role in eyeblink conditioning when using discrimination procedures involving overlapping stimuli.

Pontine stimulation overcomes developmental limitations in the neural mechanisms of eyeblink conditioning.

Pontine neuronal activation during auditory stimuli increases ontogenetically between postnatal days (P) P17 and P24 in rats. Pontine neurons are an essential component of the conditioned stimulus (CS) pathway for eyeblink conditioning, providing mossy fiber input to the cerebellum. Here we examined whether the developmental limitation in pontine responsiveness to a CS in P17 rats could be overcome by direct stimulation of the CS pathway. Eyeblink conditioning was established in infant rats on P17-P18 and P24-P25 using pontine stimulation as a CS. There were no significant age-related differences in the rate or level of conditioning. Eyeblink conditioned responses established with the stimulation CS were abolished by inactivation of the ipsilateral cerebellar nuclei and overlying cortex in both age groups. The findings suggest that developmental changes in the CS pathway play an important role in the ontogeny of eyeblink conditioning.