Developmental emergence of fear/threat learning: neurobiology, associations and timing.

Pavlovian fear or threat conditioning, where a neutral stimulus takes on aversive properties through pairing with an aversive stimulus, has been an important tool for exploring the neurobiology of learning. In the past decades, this neurobehavioral approach has been expanded to include the developing infant. Indeed, protracted postnatal brain development permits the exploration of how incorporating the amygdala, prefrontal cortex and hippocampus into this learning system impacts the acquisition and expression of aversive conditioning. Here, we review the developmental trajectory of these key brain areas involved in aversive conditioning and relate it to pups' transition to independence through weaning. Overall, the data suggests that adult-like features of threat learning emerge as the relevant brain areas become incorporated into this learning. Specifically, the developmental emergence of the amygdala permits cue learning and the emergence of the hippocampus permits context learning. We also describe unique features of learning in early life that block threat learning and enhance interaction with the mother or exploration of the environment. Finally, we describe the development of a sense of time within this learning and its involvement in creating associations. Together these data suggest that the development of threat learning is a useful tool for dissecting adult-like functioning of brain circuits, as well as providing unique insights into ecologically relevant developmental changes.

Multiple spine boutons are formed after long-lasting LTP in the awake rat.

The formation of multiple spine boutons (MSBs) has been associated with cognitive abilities including hippocampal-dependent associative learning and memory. Data obtained from cultured hippocampal slices suggest that the long-term maintenance of synaptic plasticity requires the formation of new synaptic contacts on pre-existing synapses. This postulate however, has never been tested in the awake, freely moving animals. In the current study, we induced long-term potentiation (LTP) in the dentate gyrus (DG) of awake adult rats and performed 3-D reconstructions of electron micrographs from thin sections of both axonal boutons and dendritic spines, 24 h post-induction. The specificity of the observed changes was demonstrated by comparison with animals in which long-term depression (LTD) had been induced, or with animals in which LTP was blocked by an N-methyl-D-aspartate (NMDA) antagonist. Our data demonstrate that whilst the number of boutons remains unchanged, there is a marked increase in the number of synapses per bouton 24 h after the induction of LTP. Further, we demonstrate that this increase is specific to mushroom spines and not attributable to their division. The present investigation thus fills the gap existing between behavioural and in vitro studies on the role of MSB formation in synaptic plasticity and cognitive abilities.

Altered emotional and motivational processing in the transgenic rat model for Huntington's disease.

Huntington disease (HD) is caused by an expansion of CAG repeat in the Huntingtin gene. Patients demonstrate a triad of motor, cognitive and psychiatric symptoms. A transgenic rat model (tgHD rats) carrying 51 CAG repeats demonstrate progressive striatal degeneration and polyglutamine aggregates in limbic structures. In this model, emotional function has only been investigated through anxiety studies. Our aim was to extend knowledge on emotional and motivational function in symptomatic tgHD rats. We subjected tgHD and wild-type rats to behavioral protocols testing motor, emotional, and motivational abilities. From 11 to 15 months of age, animals were tested in emotional perception of sucrose using taste reactivity, acquisition, extinction, and re-acquisition of discriminative Pavlovian fear conditioning as well as reactivity to changes in reinforcement values in a runway Pavlovian approach task. Motor tests detected the symptomatic status of tgHD animals from 11 months of age. In comparison to wild types, transgenic animals exhibited emotional blunting of hedonic perception for intermediate sucrose concentration. Moreover, we found emotional alterations with better learning and re-acquisition of discriminative fear conditioning due to a higher level of conditioned fear to aversive stimuli, and hyper-reactivity to a negative hedonic shift in reinforcement value interpreted in term of greater frustration. Neuropathological assessment in the same animals showed a selective shrinkage of the central nucleus of the amygdala. Our results showing emotional blunting and hypersensitivity to negative emotional situations in symptomatic tgHD animals extend the face validity of this model regarding neuropsychiatric symptoms as seen in manifest HD patients, and suggest that some of these symptoms may be related to amygdala dysfunction.

Alterations in synaptic curvature in the dentate gyrus following induction of long-term potentiation, long-term depression, and treatment with the N-methyl-D-aspartate receptor antagonist CPP.

Alterations in curvature of the post synaptic density (PSD) and apposition zone (AZ), are believed to play an important role in determining synaptic efficacy. In the present study we have examined curvature of PSDs and AZs 24 h following homosynaptic long-term potentiation (LTP), and heterosynaptic long-term depression (LTD) in vivo, in awake adult rats. High frequency stimulation (HFS) applied to the medial perforant path to the dentate gyrus induced LTP while HFS stimulation of the lateral perforant path induced LTD in the middle molecular layer of the dentate gyrus (DG). Curvature changes were analysed in this area using three dimensional (3-D) reconstructions of electron microscope images of ultrathin serial sections. Very large and significant changes in 3-D measurements of AZ and PSD curvature occurred 24 h following both LTP and LTD, with a flattening of the normal concavity of mushroom spine heads and a change to convexity for thin spines. An N-methyl-D-aspartate (NMDA) receptor antagonist CPP (3-[(R)-2-Carboxypiperazin-4-yl]-propyl-1-phosphonic acid) blocked the changes in curvature of mushroom and thin spine PSDs and apposition zones, actually increasing the concavity of mushroom spines as the spine engulfed the presynaptic bouton. In order to establish whether these changes resulted from the effect of the NMDA antagonist or from its coincidence with synaptic activation during testing we examined the effects of CPP alone on PSD and apposition zone curvature. It was found that CPP alone also caused a small decrease in curvature of both PSD and apposition zone of mushroom and thin spines.

The N-methyl-D-aspartate receptor antagonist CPP alters synapse and spine structure and impairs long-term potentiation and long-term depression induced morphological plasticity in dentate gyrus of the awake rat.

Long-term morphological synaptic changes associated with homosynaptic long-term potentiation (LTP) and heterosynaptic long-term depression (LTD) in vivo, in awake adult rats were analyzed using three-dimensional (3-D) reconstructions of electron microscope images of ultrathin serial sections from the molecular layer of the dentate gyrus. For the first time in morphological studies, the specificity of the effects of LTP and LTD on both spine and synapse ultrastructure was determined using an N-methyl-d-aspartate (NMDA) receptor antagonist CPP (3-[(R)-2-carboxypiperazin-4-yl]-propyl-1-phosphonic acid). There were no differences in synaptic density 24 h after LTP or LTD induction, and CPP alone had no effect on synaptic density. LTP increased significantly the proportion of mushroom spines, whereas LTD increased the proportion of thin spines, and both LTP and LTD decreased stubby spine number. Both LTP and LTD increased significantly spine head evaginations (spinules) into synaptic boutons and CPP blocked these changes. Synaptic boutons were smaller after LTD, indicating a pre-synaptic effect. Interestingly, CPP alone decreased bouton and mushroom spine volumes, as well as post-synaptic density (PSD) volume of mushroom spines.These data show similarities, but also some clear differences, between the effects of LTP and LTD on spine and synaptic morphology. Although CPP blocks both LTP and LTD, and impairs most morphological changes in spines and synapses, CPP alone was shown to exert effects on aspects of spine and synaptic structure.

LTP but not seizure is associated with up-regulation of AKAP-150.

We have used differential display to profile and compare the mRNAs expressed in the hippocampus of freely moving animals after the induction of long-term potentiation (LTP) at the perforant path-dentate gyrus synapse with control rats receiving low-frequency stimulation. We have combined this with in situ hybridization and have identified A-kinase anchoring protein of 150 kDa (AKAP-150) as a gene selectively up-regulated during the maintenance phase of LTP. AKAP-150 mRNA has a biphasic modulation in the dentate gyrus following the induction of LTP. The expression of AKAP-150 was 29% lower than stimulated controls 1 h after the induction of LTP. Its expression was enhanced 3 (50%), 6 (239%) and 12 h (210%) after induction, returning to control levels by 24 h postinduction. The NMDA receptor antagonist CPP blocked the tetanus-induced modulation of AKAP-150 expression. Interestingly, strong generalized stimulation produced by electroconvulsive shock did not increase the expression of AKAP-150. This implies that the AKAP-150 harbours a novel property of selective responsiveness to the stimulation patterns that trigger NMDA-dependent LTP in vivo. Its selective up-regulation during LTP and its identified functions as a scaffold for protein kinase A, protein kinase C, calmodulin, calcineurin and ionotropic glutamate receptors suggest that AKAP-150 encodes is an important effector protein in the expression of late LTP.

Regulated expression of the neuronal calcium sensor-1 gene during long-term potentiation in the dentate gyrus in vivo.

Neuronal calcium sensor-1 (NCS-1), the mammalian homologue of frequenin, is a member of a highly conserved family of neuron-specific calcium-binding proteins which has been implicated in exocytosis and in multiple calcium-signalling pathways, suggesting a potential involvement in mechanisms of neuronal plasticity. Here, using in situ hybridization, we report an increased induction of the mRNA encoding NCS-1 in dentate granule cells following the induction of long-term potentiation in the awake rat. We show that NCS-1 mRNA levels are increased 1 and 3 h after long-term potentiation in an N-methyl-D-aspartate receptor-dependent manner, returning to baseline expression levels by 6 h. Electroconvulsive stimulation also induced NCS-1 mRNA transcription in the dentate gyrus, but at the different time of 6 h post-seizure, returning to baseline by 12 h. These results show that regulated expression of the NCS-1 gene is part of the transcriptional response associated with activity-dependent neuronal plasticity in vivo and suggest a molecular mechanism capable of mediating a functional change in synapse sensitivity to calcium and calcium-signalling pathways after long-term potentiation.

Subfield-specific immediate early gene expression associated with hippocampal long-term potentiation in vivo.

It is not known whether NMDA receptor-dependent long-term potentiation (LTP) is mediated by similar molecular mechanisms in different hippocampal areas. To address this question we have investigated changes in immediate early gene and protein expression in two hippocampal subfields following the induction of LTP in vivo and in vitro. In granule cells of the dentate gyrus, LTP induced in vivo by tetanic stimulation of the perforant path was followed by strong induction of the immediate early genes (IEGs) Zif268, Arc and Homer. The increase in Zif268 mRNA was accompanied by an increase in protein expression. In contrast, we were unable to detect modulation of the IEGs Zif268, Arc, Homer and HB-GAM following induction of LTP by high-frequency stimulation of the commissural projection to CA1 pyramidal cells in vivo. In this pathway, we also failed to detect modulation of Zif268 protein levels. Zif268, Arc and Homer can be modulated in CA1 pyramidal cells approximately twofold after electroshock-induced maximal seizure, which demonstrates potential responsiveness to electrical stimuli. When LTP was induced in vitro neither CA1 pyramidal cells nor granule cells showed an increase in Zif268, Arc or Homer mRNA. However, in the slice preparation, granule cells have a different transcriptional state as basal IEG levels are elevated. These results establish the existence of subfield-specific transcriptional responses to LTP-inducing stimulation in the hippocampus of the intact animal, and demonstrate that in area CA1-enhanced transcription of Zif268, Arc and Homer is not required for the induction of late LTP.

Age-related modifications of contextual information processing in rats: role of emotional reactivity, arousal and testing procedure.

Two experiments were conducted to examine contextual information processing in adult (7 months) and aged (22 months) Wistar rats. In Experiment 1, rats were tested for contextual fear conditioning when exposed to six series, one per day, of ten pairings of a tone (CS) with a foot-shock (US) delivered in one of a two-compartment apparatus. Conditioned fear was estimated by recording: (1) the amount of freezing in the shock compartment; and (2) the time spent avoiding the shock compartment. Results show that, after only one series of ten CS-US pairings, all rats showed freezing in the shock compartment, with aged rats exhibiting the stronger response. Adult rats also avoided the shock compartment during place preference tests in contrast to aged rats, that spent an equivalent time - with an intense freezing reaction - in both the shock and the safe compartments. After 60 CS-US pairings, contextual freezing in the shock compartment decreased in both groups, but, contrary to adults, aged rats were still not avoiding that compartment. In Experiment 2, radial maze performance was studied under distinct quantitative extra-maze cueing conditions (poor versus rich) and successive context shifts. Compared to adults, aged rats were impaired when trained initially under poor cueing conditions. No group difference was evident when rats were transferred to a context involving more cues (rich cueing conditions), but age-related impairments re-emerged when rats were returned to the original poor cueing conditions. Thus, the fact that performance deficits in a given task were restricted to certain testing procedures suggests that aging affects more the utilization than the processing of contextual information.

Effects of lesions to the hippocampus on contextual fear: evidence for a disruption of freezing and avoidance behavior but not context conditioning.

The effects of ibotenic lesions of the hippocampus on conditioning to contextual cues during classical fear conditioning in rats were evaluated by (a) the amount of freezing elicited by contextual cues and (b) the relative avoidance of a shock compartment. In Experiment 1, lesions to the hippocampus had no effect on contextual freezing and marginally affected avoidance after repeated sessions. Experiment 2 showed that lesions to the hippocampus disrupted avoidance when tested after a single conditioning session, while leaving unaffected the acquisition of contextual freezing. Experiment 3 indicated that these lesions decreased the acquisition of contextual freezing when higher footshock intensity was used but had no effect on avoidance after repeated conditioning sessions. These results show that freezing and avoidance do not quantify context conditioning similarly. They further indicate that lesions to the hippocampus may disrupt the expression of these behaviors used as measures of context conditioning but not the acquisition of context conditioning per se.

Heterosynaptic LTD and depotentiation in the medial perforant path of the dentate gyrus in the freely moving rat.

We examined the characteristics of heterosynaptic long-term depression (LTD) and depotentiation of previously established long-term potentiation (LTP) in the medial and lateral entorhinal afferents to the dentate gyrus in the awake rat. Rats were prepared for chronic recording of dentate gyrus evoked potentials to activation of the medial and lateral perforant paths. This study in awake rats confirms that heterosynaptic LTD can be induced at inactive medial perforant path synapses in conjunction with the induction of LTP produced by high-frequency stimulation of the lateral perforant path. This form of LTD was long lasting and reversible by tetanic stimulation delivered to the depressed pathway. In contrast, tetanic stimulation of the medial perforant path had only a small heterosynaptic effect on the lateral pathway, suggesting that the two input pathways to the dentate gyrus are not symmetrical in their ability to induce heterosynaptic LTD. We also examined the ability of high-frequency stimulation of one pathway to produce depotentiation of the other pathway. We found that when LTP was first induced in the medial perforant path, depotentiation was induced heterosynaptically by tetanization of the lateral pathway. Both newly established LTP (30 min) and LTP induced and saturated by repeated tetanic stimulation over several days can be depotentiated heterosynaptically. Moreover, depotentiation of the medial perforant path synapses was found to be linearly correlated with the magnitude of LTP induced in the lateral perforant path synapses, and subsequent tetanic stimulation of the depotentiated medial perforant path restored LTP to an extent that counterbalanced depotentiation. The saturation and repotentiation experiments provide clear support for the conclusion that the rapid reversal of LTP reflects true depotentiation of the medial input. Again, as with heterosynaptic LTD, tetanization of the medial perforant path had little effect on previously induced LTP in the lateral path. These results provide evidence that medial perforant path synapses can be depressed and depotentiated heterosynaptically. They suggest that in the intact rat synaptic changes in the afferents to the dentate gyrus from the lateral entorhinal cortex exert powerful control over ongoing or recent synaptic plasticity in the medial entorhinal afferents.

Low-frequency trains of paired stimuli induce long-term depression in area CA1 but not in dentate gyrus of the intact rat.

We have examined the efficacy of a recently introduced protocol for inducing homosynaptic long-term depression (LTD) in area CA1 of the anesthetized rat (Thiels et al. [1994] J Neurophysiol 72:3009-3116.). In area CA1 of the awake animal, this protocol, consisting of 200 pairs of pulses delivered at 0.5 Hz, with an interpulse interval of 25 ms, consistently produced LTD, provided the initial pulse was sufficiently strong to produce significant paired-pulse depression of the evoked response. We extended these experiments to the dentate gyrus, using either paired pulses given to the perforant path in the awake adult rat, or, in the anesthetized adult, a two-pathway pairing procedure, in which the first pulse was delivered to the commissural input to the dentate gyrus and the second to the perforant path. In both cases, the first pulse led to substantial suppression of the response evoked by the second pulse. With neither protocol, however, was there any evidence for LTD or depotentiation. Paired-pulse stimulation of the perforant path of young rats (10-11 days) also failed to induce LTD or depotentiation of the population excitatory postsynaptic potential (EPSP). Thus, the dentate gyrus in the intact animal appears to be less susceptible to LTD and depotentiation than area CA1, a conclusion consistent with previous experiments in which we found that stimulation at 1-5 Hz produced LTD/depotentiation in area CA1 of young (but not adult) rats in vivo but was ineffective at any age in the dentate gyrus. Our results do not rule out the possibility that other, untested protocols may produce homosynaptic LTD and/or depotentiation in the dentate gyrus in vivo.

Stimulation at 1-5 Hz does not produce long-term depression or depotentiation in the hippocampus of the adult rat in vivo.

1. We examined the efficacy of low-frequency trains (1-5 Hz) in producing long-term depression (LTD) or depotentiation in the hippocampus of the awake adult rat and in anesthetized rats aged from 10 days to 3 mo. 2. In the dentate gyrus we found no evidence that low-frequency trains produce either depotentiation or LTD in the awake, adult animal or in the anesthetized animal at any age tested (10 days-adult). 3. In area CA1 of both awake and anesthetized adult rats, we also found no evidence that low-frequency trains induced either LTD or depotentiation. Only in area CA1 of very young rats (10-11 days) was clear evidence for LTD and depotentiation obtained; at this age experiments were only possible in anesthetized animals. By 16 days, the ability to display both LTD and depotentiation was lost. 4. These experiments suggest that repetitive low-frequency stimulation evokes a developmentally regulated form of activity-dependent depression that in the hippocampus is limited to specific pathways in the young animal. Our results leave open the question of whether alternative patterns of activity can induce LTD and/or depotentiation in the adult awake rat.

Potentiation or depression of synaptic efficacy in the dentate gyrus is determined by the relationship between the conditioned and unconditioned stimulus in a classical conditioning paradigm in rats.

Learning a conditioned stimulus (CS)-unconditioned stimulus (US) association is accompanied by a variety of long-lasting changes in physiology and chemistry of the synapse in the dentate gyrus. To determine the time course of synaptic modification during learning, changes in the perforant path-dentate gyrus-evoked field potentials were measured in rats performing a classical conditioning (paired tone and footshock) or pseudoconditioning (unpaired tone and footshock) task. Over the course of 4 days of training, differential changes in the evoked response were observed in the two groups. In the conditioned group, there was an increase in the slope of the excitatory postsynaptic potential (EPSP) which started after five tone-shock paired trials and lasted for more than 40 min, outlasting the training session by 20 min. In contrast, a decrease in the slope of the EPSP which commenced after training and lasted for at least 1 h was observed in the pseudoconditioned group. In both groups there was a prolonged decrease in the amplitude of the population spike. The increase in the EPSP was reduced and the duration tended to shorten over days of training in the conditioned group, whereas in the pseudoconditioned group the decrease in the EPSP tended to increase. Off-line analysis of suppression of lever-pressing for food reward during the presentation of the tone, indicated that the conditioned rats had learned the tone-footshock association. Temperature was measured in the dentate gyrus of rats undergoing an identical procedure. In both groups slight temperature increases were observed, with no difference in amplitude and time-course between the groups. The differential effect of conditioning and pseudoconditioning on the evoked response and changes in temperature eliminate the possibility that effects of stress, arousal and muscular effort are the primary cause of the changes in the EPSP. The results suggest that behavioural events can exert bidirectional control of synaptic strength of entorhinal cortex inputs to the dentate gyrus and that the sign of synaptic modification is at least in part determined by the temporal relationship between these events. The data are discussed in terms of the type of neural activity that may mediate the processing of information in the dentate gyrus.

Long-term potentiation of hippocampal afferents and efferents to prefrontal cortex: implications for associative learning.

It has been proposed that the physical substrate of memory resides in alterations of the strengths or weights of modifiable synaptic connections. In recent years, the hypothesis that the mechanisms underlying a particular form of synaptic plasticity, known as long-term potentiation, or LTP, are activated during learning and may actually subserve the formation of associative memories, has gained much empirical support. This paper reviews experimental studies suggesting that changes in synapse physiology and chemistry are involved in the formation of neural associative representation in hippocampal networks during classical conditioning. Recent experiments investigating LTP and learning-induced synaptic changes at hippocampal outputs to the prefrontal cortex are reported. The results provide a working framework within which the dynamics of information storage in hippocampal and prefrontal cortical networks is profiled.

Linear relationship between the maintenance of hippocampal long-term potentiation and retention of an associative memory.

The hypothesis that the maintenance or decay of an associative memory trace after an extended retention interval is a function of the residual strength of the synapses originally strengthened during learning was examined in a classical conditioning paradigm in which high-frequency stimulation of a hippocampal input--the medial perforant path--served as a conditioned stimulus. Rats received perforant path stimulus-foot shock pairings while engaged in a previously acquired food-motivated lever-pressing task. Conditioned suppression of lever pressing was the behavioral measure of learning and retention of the association. Stimulus trains to the perforant path at an intensity above the threshold for eliciting a population spike induced long-term potentiation of synaptic transmission in the dentate gyrus. Synaptic potentials recorded extracellularly in the dentate gyrus were subsequently monitored for 31 days to examine quantitatively the decay of synaptic potentiation, a period after which retention of the learned association was assessed. All rats learned the association to a similar extent and displayed equivalent amounts of long-term potentiation by the end of conditioning. A slowly decaying function of synaptic potentiation was observed in remembering rats, i.e., rats with high retention performance after the 31-day learning-to-retention interval, while forgetting was associated with a rapid decay of long-term potentiation. Behavioral performance at the long-term memory test was linearly correlated with the amplitude of long-term potentiation maintained just prior to the retention test. The results favor the hypothesis that long-term associative memory depends, at least in part, on the maintenance of elevated synaptic strengths in the pathway activated during learning and suggest a role for the lasting component of long-term potentiation in the maintenance of memory.

Linear relation between the magnitude of long-term potentiation in the dentate gyrus and associative learning in the rat. A demonstration using commissural inhibition and local infusion of an N-methyl-D-aspartate receptor antagonist.

Field potentials were recorded in the dentate gyrus of freely-moving rats in a classical conditioning paradigm in which high-frequency stimulation of the perforant path served as a conditioned stimulus. Paired or unpaired perforant path stimulus-footshock presentations were given to animals engaged in a previously acquired food-motivated lever-pressing task. Conditioned suppression of lever-pressing was the behavioural measure of conditioning. Perforant path stimulus trains at an intensity above spike threshold induced long-term potentiation of synaptic transmission in the dentate gyrus. In this condition, animals learned the perforant path stimulus-shock association. Three strategies were employed to block the induction or reduce the magnitude of long-term potentiation induced by the conditioned stimulus: (1) reduction of the intensity of the stimulus below the spike threshold resulted in no long-term potentiation and a failure by the animals to learn the perforant path stimulus-shock association; (2) inhibitory modulation of long-term potentiation by high-frequency activation of commissural input to the dentate gyrus resulted in learning deficits; (3) chronic infusion of DL-2-amino-5-phosphonovalerate, a selective antagonist of the N-methyl-D-aspartate subtype of glutamate receptor, blocked the induction of long-term potentiation and prevented associative learning. A highly significant linear relation emerged from a correlational analysis between the magnitude of the change in synaptic efficacy at the activated synapses and the amount the animals learned about the perforant path stimulus-shock association. The results presented in this paper are consistent with the hypothesis that associative learning depends on the development of lasting changes in synaptic function. We propose that the activation of N-methyl-D-aspartate receptors in the dentate gyrus is involved in this process and that the more change in synaptic efficacy is produced in the activated network, the more the animals learn.