Aging and learning-specific changes in single-neuron activity in CA1 hippocampus during rabbit trace eyeblink conditioning.

Rabbit trace eyeblink conditioning is a hippocampus-dependent task in which the auditory conditioned stimulus (CS) is separated from the corneal airpuff unconditioned stimulus (US) by a 500-ms empty trace interval. Young rabbits are able to associate the CS and US and acquire trace eyeblink conditioned responses (CRs); however, a subset of aged rabbits show poor learning on this task. Several studies have shown that CA1-hippocampal activity is altered by aging; however, it is unknown how aging affects the interaction of CA1 single neurons within local ensembles during learning. The present study examined the extracellular activity of CA1 pyramidal neurons within local ensembles in aged (29-34 mo) and young (3-6 mo) rabbits during 10 daily sessions (80 trials/session) of trace eyeblink conditioning. A single surgically implanted nonmovable stereotrode was used to record ensembles ranging in size from 2 to 12 separated single neurons. A total of six young and four aged rabbits acquired significant levels of CRs, whereas five aged rabbits showed very few CRs similar to a group of five young pseudoconditioned rabbits. Pyramidal cells (2,159 total) were recorded from these four groups during training. Increases in CA1 pyramidal cell firing to the CS and US were diminished in the aged nonlearners. Local ensembles from all groups contained heterogeneous types of pyramidal cell responses. Some cells showed increases while others showed decreases in firing during the trace eyeblink trial. Hierarchical clustering was used to isolate seven different classes of single-neuron responses that showed unique firing patterns during the trace conditioning trial. The proportion of cells in each group was similar for six of seven response classes. Unlike the excitatory modeling patterns reported in previous studies, three of seven response types (67% of recorded cells) exhibited some type of inhibitory decrease to the CS, US, or both. The single-neuron response classes showed different patterns of learning-related activity across training. Several of the single-neuron types from the aged nonlearners showed unique alterations in response magnitude to the CS and US. Cross-correlation analyses suggest that specific single-neuron types provide more correlated single-neuron activity to the ensemble processing of information. However, aged nonlearners showed a significantly lower level of coincident pyramidal cell firing for all cell types within local ensembles in CA1.

Remodeling of hippocampal synapses after hippocampus-dependent associative learning.

The aim of this study was to determine whether hippocampus-dependent associative learning involves changes in the number and/or structure of hippocampal synapses. A behavioral paradigm of trace eyeblink conditioning was used. Young adult rabbits were given daily 80 trial sessions to a criterion of 80% conditioned responses in a session. During each trial, the conditioned (tone) and unconditioned (corneal airpuff) stimuli were presented with a stimulus-free or trace interval of 500 msec. Control rabbits were pseudoconditioned by equal numbers of random presentations of the same stimuli. Brain tissue was taken for morphological analyses 24 hours after the last session. Synapses were examined in the stratum radiatum of hippocampal subfield CA1. Unbiased stereological methods were used to obtain estimates of the total number of synapses in this layer as well as the area of the postsynaptic density. The data showed that the total numbers of all synaptic contacts and various morphological subtypes of synapses did not change in conditioned animals. The area of the postsynaptic density, however, was significantly increased after conditioning in axospinous nonperforated synapses. This structural alteration may reflect an addition of signal transduction proteins (such as receptors and ion channels) and the transformation of postsynaptically silent synapses into functional ones. The findings of the present study indicate that cellular mechanisms of hippocampus-dependent associative learning include the remodeling of existing hippocampal synapses. Further studies examining various time points along the learning curve are necessary to clarify the issue of whether these mechanisms also involve the formation of additional synaptic contacts.

Neurotoxic lesions of the dorsal hippocampus disrupt auditory-cued trace heart rate (fear) conditioning in rabbits.

The first experiment in this study used the classical heart rate (HR) conditioning paradigm to determine if rabbits could associate an auditory conditioned stimulus (CS) and a fear-producing shock-unconditioned stimulus (US) separated by an empty 10-s trace interval. Trace conditioned rabbits (n = 7) acquired significant bradycardiac conditioned HR responses on CS-alone test trials during a single 35-trial conditioning session. Control animals (n = 7) which received unpaired CSs and USs did not show HR conditioning. During a retention session of CS-alone trials 24 h after the conditioning session, some trace-conditioned animals showed conditioned HR responses immediately following CS onset (n = 3), while others showed responses appropriately timed to the US onset (n = 4) used in trace conditioning 24 h earlier. Thus, rabbits remember the duration of the long 10-s trace interval 24 h after a single day of training. The second part of this study sought to determine if cells in the dorsal hippocampus play a role in trace HR conditioning. Rabbits were given bilateral ibotenic acid lesions in the neocortex (n = 7) or dorsal hippocampus (n = 8). During trace conditioning and retention, neocortical animals showed conditioned HR responses to the CS, whereas the hippocampal group showed no significant HR conditioning. One week after trace conditioning, the same animals received a delay HR conditioning session where no trace interval separated the CS and US. During delay conditioning, hippocampal animals showed significant conditioned HR responses to the CS that were similar to the neocortical group. Thus, the dorsal hippocampus plays a critical role in rabbit HR conditioning when the CS and US are separated by a 10-s trace interval. This paradigm may be ideal for in vivo electrophysiological recording studies because rabbits are easily immobilized during the testing procedure, and learning occurs during a single day of training.

Cortical involvement in acquisition and extinction of trace eyeblink conditioning.

Previous studies have implicated 2 cortical regions interconnected with the hippocampal formation, the retrosplenial cortex (RSC) and the medial prefrontal cortex (mPFC), as loci important for the acquisition of hippocampally dependent trace eyeblink conditioning. These loci have also been proposed to serve as long-term storage sites of task critical information. This study used lesions made prior to training to investigate the roles of the RSC, as well as the caudal and rostral subdivisions of the mPFC, in the acquisition and subsequent extinction of trace eyeblink conditioning in the rabbit. The caudal mPFC and rostral mPFC were shown to be critical for acquisition and extinction of the conditioned reflex, respectively. The data indicate that the RSC is not critical for acquisition or extinction of the trace conditioned reflex.

Hippocampal encoding of non-spatial trace conditioning.

Trace eyeblink classical conditioning is a non-spatial learning paradigm that requires an intact hippocampus. This task is hippocampus-dependent because the auditory tone conditioned stimulus (CS) is temporally separated from the corneal airpuff unconditioned stimulus (US) by a 500-ms trace interval. Our laboratory has performed a series of neurophysiological experiments that have examined the activity of pyramidal cells in the CA1 area of the hippocampus during trace eyeblink conditioning. We have found that the non-spatial stimuli involved in this paradigm are encoded in the hippocampus in a logical order that is necessary for their association and the subsequent expression of behavioral learning. Although there were many profiles of single neurons responding to the CS-US trial during training, the majority of the neurons showed an increase in activity to the airpuff-US. Prior to learning, it appears that hippocampal cells and ensembles of cells were preferentially attending to the stimulus with immediate behavioral importance, the US. Hippocampal cells then began to respond to the associated neutral stimulus, the CS. Shortly thereafter, animals began to show increases in the behavioral expression of CRs. In some experiments, hippocampal neurons from aged animals exhibited impairments in the encoding of CS and US information. These aged animals were not able to associate these stimuli and acquire trace eyeblink CRs. Our findings along with the findings of other spatial learning studies, suggest that the hippocampus is involved in encoding information about discontiguous sets of stimuli, either spatial or nonspatial, especially early in the learning process.

Hippocampectomy disrupts auditory trace fear conditioning and contextual fear conditioning in the rat.

The hippocampus is believed to be an important structure for learning tasks that require temporal processing of information. The trace classical conditioning paradigm requires temporal processing because the conditioned stimulus (CS) and the unconditioned stimulus (US) are temporally separated by an empty trace interval. The present study sought to determine whether the hippocampus was necessary for rats to perform a classical trace fear conditioning task in which each of 10 trials consisted of an auditory tone CS (1 5-s duration) followed by an empty 30-s trace interval and then a fear-producing floor-shock US (0.5-s duration). Several weeks prior to training, animals were anesthetized and given aspiration lesions of the neocortex (NEO; n = 6), hippocampus and overlying neocortex (HIPP; n = 7), or no lesions at all (control; n = 6). Approximately 24 h after trace conditioning, NEO and control animals showed a significant decrease in movement to a CS-alone presentation that was indicative of a conditioned fear response. Animals in the HIPP group did not show conditioned fear responses to the CS alone, nor did a pseudoconditioning group (n = 7) that was trained with unpaired CSs and USs. Furthermore, all groups except the HIPP group showed conditioned fear responses to the original context in which they received shock USs. One week later, HIPP, NEO, and control animals received delay fear-conditioning trials with no trace interval separating the CS and US. Six of seven HIPP animals could perform the delay version, but none could perform the trace version. This result suggests that the trace fear task is a reliable and useful model for examining the neural mechanisms of hippocampally dependent learning.

Sequence of single neuron changes in CA1 hippocampus of rabbits during acquisition of trace eyeblink conditioned responses.

The sequence of changes in single neuron activity in the CA1 area of the rabbit hippocampus was examined during daily sessions (80 trials/session) of hippocampally dependent nonspatial trace eyeblink (i.e., nictitating membrane response) conditioning. Each trial for trace conditioned animals (n = 7) consisted of a tone conditioned stimulus (CS; 6 kHz; 90 dB, 100 ms) followed by a 500-ms silent trace period, then a corneal airpuff unconditioned stimulus (US; 3.0 psi; 150 ms). Control animals (n = 5) received unpaired CSs and USs. Most pyramidal (n = 309) and theta (n = 21) cells were recorded for a single day of training. The activity of cells for each animal were grouped according to: the day of training that CRs began to increase and the day of training that CR performance became asymptotic. Pyramidal cells from trace conditioned animals demonstrated several stages of learning-related activity: large increases in activity after both the CS and US early in conditioning on the day of training when CRs began to increase, smaller moderate increases in activity on the following days of training, and decreases in activity after the US during asymptotic CRs. Pyramidal cell-increases declined significantly across the trials of each daily session. Theta cells showed an activity pattern opposite to the pyramidal cells, consistent with the notion that theta cells have an inhibitory influence on pyramidal cells. Single pyramidal cells also were categorized into response profiles. Most pyramidal response profiles showed increases in activity specific to the day of initial CRs. Two of the pyramidal response profiles may be involved in assessing the temporal properties of the CS-US trace conditioning trial.

Changes of synaptic efficacy in the medial geniculate nucleus as a result of auditory classical conditioning.

In this study we examined inputs to neurons in the medial subnucleus of the medial geniculate nucleus (mMG) for changes of synaptic efficacy associated with heart-rate conditioning to an auditory conditioned stimulus (CS). Conditioning-related changes of synaptic efficacy were measured in awake animals by examining mMG single-unit responses evoked by stimulation of one of two areas that send auditory CS and nonauditory information monosynaptically to the mMG, the brachium of the inferior colliculus (BlC) and the superior colliculus (SC). Synaptic efficacy was measured before, immediately after, and 1 hr after one session of classical conditioning with a tone CS and a corneal airpuff unconditioned stimulus. To determine whether conditioning produced changes of synaptic efficacy on the auditory BlC inputs to mMG cells and not general changes of cellular excitability, analyses of synaptic efficacy were performed on the mMG units that exhibited short-latency evoked responses (< 3.5 msec) to both BlC and SC stimulation. Analyses revealed that the BlC but not the SC test stimulus-evoked unit activity from the same neurons exhibited the following changes immediately after conditioning: decreases in unit response latency, increases in unit response reliability, and increases in spike frequency. BlC-evoked unit responses after pseudoconditioning did not exhibit these changes in unit responding. These results suggest that the synapses carrying auditory CS information to mMG neurons increase in strength as the result of associative conditioning with an acoustic CS. Some of these changes of synaptic efficacy remained 1 hr after training.

Immunohistochemical expression of the c-Fos protein in the spinal trigeminal nucleus following presentation of a corneal airpuff stimulus.

This study examined the expression of the c-Fos protein in the rabbit's central nervous system to determine which areas are activated by the presentation of a corneal airpuff. Previous work has shown that pairing a corneal airpuff unconditioned stimulus (US) with a tone conditioned stimulus (CS) produces reliable heart rate (HR) conditioning. In this study restrained awake rabbits received 100 corneal airpuffs. Brains were then processed immunohistochemically for the c-Fos protein. In animals that received the airpuff the ventral portion of the ipsilateral spinal trigeminal subnucleus caudalis (SVc) and interpolaris (SVi), and the dorsal raphe nucleus exhibited a greater number of c-Fos labeled cells compared to control animals. Another group of animals was given microinjections of WGA-HRP in the medial nucleus of the medial geniculate (mMG) to determine if this critical auditory area of the HR conditioning circuitry receives projections from SVc and SVi. These injections produced retrograde labeling in the same areas of SVc and SVi activated by the airpuff. Thus, a corneal airpuff activates neurons in SVc and SVi which could then activate neurons in mMG. This provides additional evidence that CS and US information converge in mMG.

Simultaneous single unit recording in the medial nucleus of the medial geniculate nucleus and amygdaloid central nucleus throughout habituation, acquisition, and extinction of the rabbit's classically conditioned heart rate.

The present study examined single neuron activity in the medial nucleus of the medial geniculate (mMG) and amygdaloid central nucleus (ACe) simultaneously across several phases of differential heart rate conditioning (habituation, acquisition, and extinction). Within the same animals, the magnitude of mMG and ACe unit responses to two tone conditioned stimuli (CS) exhibited habituation, differential acquisition, and extinction. Neurons in each area developed a differential response latency to the CSs during acquisition, suggesting that mMG and ACe may be involved in changes of synaptic efficacy. Units in both areas rapidly developed a differential response magnitude to the CSs (< 6 acquisition trials), however, mMG units responded to the CSs with a shorter latency than ACe units across all phases of training. This suggests that unlearned and learned CS information may access mMG before ACe. These results are consistent with the notion that conditioning-induced plasticity which occurs in mMG may influence the conditioning-induced plasticity that occurs further downstream in the amygdala.

Destruction of neurons in the VPM thalamus prevents rabbit heart rate conditioning.

The present study examined the role of the ventral posterior medial nucleus of the thalamus (VPM) in classical heart rate (HR) conditioning using an acoustic conditioned stimulus (CS) and a corneal air puff unconditioned stimulus (US). Previous research suggests that VPM neurons are activated during the presentation of a corneal air puff US. Rabbits were given ibotenic acid lesions in the VPM and subjected to one Pavlovian HR conditioning session. The results of the present study demonstrate that destruction of cell bodies in the VPM reduces HR conditioning to the level of a pseudoconditioning control without affecting HR baseline, or orienting responses to the CS. Lesions of the VPM also significantly augment the tachycardiac unconditioned response, suggesting that VPM lesions alter the somatosensory processing of the US.

Electrolytic and ibotenic acid lesions of the medial subnucleus of the medial geniculate prevent the acquisition of classically conditioned heart rate to a single acoustic stimulus in rabbits.

The present study examined the role of the medial subnucleus of the medial geniculate (mMG) in classical heart rate (HR) conditioning to a single acoustic conditioned stimulus (CS) in rabbits. Previous electrophysiological and neuroanatomical studies have implicated the mMG as a potential site of plasticity in forming the HR conditioned response (CR) to acoustic stimuli. In addition, several studies have found that bilateral lesions of the rabbit mMG prevent differential conditioning to acoustic stimuli, however animals still exhibit a significant bradycardiac response to the tones. In order to determine if the residual bradycardia seen in differential conditioning studies was due to learned responses or non-associative effects, rabbits with either bilateral electrolytic or ibotenic acid lesions of mMG, and animals with lesions outside of mMG (lesion control), were subjected to one session of single tone Pavlovian conditioning. In this paradigm, an acoustic CS was paired with an aversive unconditioned stimulus (US) in the conditioning groups, and in a pseudoconditioning group the CS and US were unpaired. The results suggest that bilateral lesions of mMG prevent the acquisition of the HR CR relative to control lesioned animals. The results also suggest that cells intrinsic to mMG are involved in conditioned bradycardia to a single tone, as well as in the discrimination between two tones, as reported previously. The lesion effects upon CRs are discussed with respect to other areas in the acoustic thalamus.

Air puff versus shock unconditioned stimuli in rabbit heart rate conditioning.

The present study examined whether corneal air puff can be used as an unconditioned stimulus to elicit reliable classically conditioned heart rate (HR) responses in rabbits. The conditioned and unconditioned HR responses were assessed during Pavlovian conditioning with different intensities of paraorbital shock (2.7, 1.2, or 0.5 mA) or corneal air puff (18.3, 5.9, or 2.2 N/cm2) unconditioned stimuli (UCSs). Each experimental group was given one acquisition session during which an acoustic conditioned stimulus was paired with either the high, medium, or low intensity of a shock or air puff UCS. The results suggest that: a) HR is reliably conditioned with a high-intensity air puff UCS, and with medium- or high-intensity paraorbital shock stimuli; and b) only UCSs that elicit a tachycardiac unconditioned HR response reliably support HR conditioning. It was concluded that either air puff or paraorbital shock can serve as an effective UCS for HR conditioned responses.

Morphine's effects on differential serial compound conditioning and reflex modification of the rabbit's (Oryctolagus cuniculus) nictitating membrane response.

This study sought to determine the effects of morphine (0, 2, and 5 mg/kg) on (a) differential classical conditioning of the rabbit's (Oryctolagus cuniculus) nictitating membrane response (NMR) to the serial compounds A-X-unconditioned stimulus (US) and B-X-US (Experiment 1) and (b) the reflex modification effects of the compounds and their components (Experiment 2). These experiments determined specifically morphine's effects on the distinctiveness and time course of stimulus representations by examining morphine's dose-response effect on (a) differential responding to A and B and their conditional control over responding to X within the compounds and (b) the unconditioned excitatory effects of the compounds and their components as assessed by their ability to modify the amplitude of the unconditioned NMR. The results of these experiments indicate that morphine, in a dose-dependent manner, can operate to profoundly attenuate the distinctiveness and persistence (short-term memory) of stimulus representations.