Mouse models of impaired fear memory exhibit deficits in amygdalar LTP

Inbred mouse strains have different genetic backgrounds that can result in impairments of synaptic plasticity and memory. They are valuable models for probing the mechanisms of memory impairments. We examined fear memory in several inbred strains, along with synaptic plasticity that may underlie fear memory. Long-term potentiation (LTP) is a form of activity-dependent synaptic plasticity that is a candidate cellular mechanism for some forms of learning and memory. Strains with impaired contextual or cued fear memory may have selective LTP deficits in different hippocampal subregions, or in the amygdala. We measured fear memory and its extinction in five inbred strains: C57BL/6NCrlBR (B6), A/J, BALB/cByJ (BALB), C57BL/10J (B10), and SM/J (SM). We also measured LTP in the basolateral amygdala and in the hippocampal Schaeffer collateral-commissural (SC) and medial perforant pathways (MPP). All strains exhibited intact contextual fear memory 24 h post-training, but cued fear memory was impaired in strains A/J, BALB, and SM. At 1 h post-training, both contextual and cued fear memory deficits were more widespread: all strains except for B6 and B10 showed impairments of both types of memory. Contextual fear extinction was impaired in BALB and SM. We found that amygdalar LTP was reduced in strains A/J and BALB, but SC LTP was intact in all strains (except for a selective multi-train LTP impairment in BALB). MPPLTP was similar in all five strains. Thus, reduced amygdalar LTP is correlated with impaired cued fear memory in strains A/J and BALB. Also, hippocampal SC LTP is more strongly correlated with 24-h (long-term) than with 1-h (short-term) contextual fear memory. In this first conjoint study of amygdala-dependent memory and amygdalar LTP in inbred mice, we identified specific hippocampal and amygdalar LTP deficits that correlate with fear memory impairments. These deficits should be considered when selecting inbred strains for genetic modification.     

Fear learning induces persistent facilitation of amygdala synaptic transmission

In the maintenance phase of fear memory, synaptic transmission is potentiated and the stimulus requirements and signalling mechanisms are altered for long-term potentiation (LTP) in the cortico-lateral amygdala (LA) pathway. These findings link amygdala synaptic plasticity to the coding of fear memories. Behavioural experiments suggest that the amygdala serves to store long-term fear memories. Here we provide electrophysiological evidence showing that synaptic alterations in rats induced by fear conditioning are evident in vitro 10 days after fear conditioning. We show that synaptic transmission was facilitated and that high-frequency stimulation dependent LTP (HFS-LTP) of the cortico-lateral amygdala pathway remained attenuated 10 days following fear conditioning. Additionally, we found that the low-frequency stimulation dependent LTP (LFS-LTP) measured 24 h after fear conditioning was absent 10 days post-training. The persistent facilitation of synaptic transmission and occlusion of HFS-LTP suggests that, unlike hippocampal coding of contextual fear memory, the cortico-lateral amygdala synapse is involved in the storage of long-term fear memories. However, the absence of LFS-LTP 10 days following fear conditioning suggests that amygdala physiology 1 day following fear learning may reflect a dynamic state during memory stabilization that is inactive during the long-term storage of fear memory. Results from these experiments have significant implications regarding the locus of storage for maladaptive fear memories and the synaptic alterations induced by these memories.     

Long-term potentiation in the amygdala: a mechanism for emotional learning and memory

In the mammalian brain, LTP is an enduring form of synaptic plasticity that is posited to have a role in learning and memory. Compelling new evidence for this view derives from studies of LTP in the amygdala, a brain structure that is essential for simple forms of emotional learning and memory, such as Pavlovian fear conditioning in rats. More specifically, antagonists of the NMDA receptor block both amygdaloid LTP induction and fear conditioning, fear conditioning induces increases in amygdaloid synaptic transmission that resemble LTP, and genetic modifications that disrupt amygdaloid LTP eliminate fear conditioning. Collectively, these results provide the most-convincing evidence to date that LTP mediates learning and memory in mammals.     

Amygdala, long-term potentiation, and fear conditioning.

Fear conditioning, during which emotional significance is attached to an initially biologically insignificant conditioned stimulus, when such neutral stimulus is paired with an aversive unconditioned stimulus, provides an experimental paradigm that is most commonly used to study fear learning. The amygdala, a sub-cortical nuclear group, is a brain structure critically important for fear conditioning. Recent studies indicate that both fear conditioning-induced neuronal plasticity and LTP at the amygdala synapses share common mechanisms of induction and expression. These findings provide the most direct evidence yet available that the mechanisms of LTP are recruited in the experimental animals during behavioral training and that such mechanisms might be utilized for memory storage.     

Fear memories induce a switch in stimulus response and signaling mechanisms for long-term potentiation in the lateral amygdala

Activity-dependent modification of synapses is fundamental for information storage in the brain and underlies behavioral learning. Fear conditioning is a model of emotional memory and anxiety that is expressed as an enduring increase in synaptic strength in the lateral amygdala (LA). Here we analysed synaptic plasticity in the rat cortico-LA pathway during maintenance of fear memory. We show for the first time that the stimulus frequency for synaptic potentiation is switched during maintenance of fear memory, and the underlying signaling mechanisms are altered in the cortico-LA pathway. In slices from fear-conditioned animals, high-frequency stimulation-induced (HFS) long-term potentiation (LTP) was attenuated, whereas low-frequency stimulation (LFS) elicited a long-lasting potentiation. HFS generates robust LTP that is dependent on N-methyl-d-aspartate receptor (NMDAR) and L-type voltage-gated calcium channel (VGCC) activation in control animals, whereas in fear-conditioned animals HFS LTP is NMDAR- and VGCC-independent. LFS-LTP is partially NMDAR-dependent, but VGCCs are necessary for potentiation in fear memory. Collectively, these results show that during maintenance of fear memory the stimulus requirements for amygdala afferents and critical signaling mechanisms for amygdala synaptic potentiation are altered, suggesting that cue-engaged synaptic mechanisms in the amygdala are dramatically affected as a result of emotional learning.     

Memory consolidation of Pavlovian fear conditioning: a cellular and molecular perspective

Pavlovian fear conditioning has emerged as a leading behavioral paradigm for studying the neurobiological basis of learning and memory. Although considerable progress has been made in understanding the neural substrates of fear conditioning at the systems level, until recently little has been learned about the underlying cellular and molecular mechanisms. The success of systems-level work aimed at defining the neuroanatomical pathways underlying fear conditioning, combined with the knowledge accumulated by studies of long-term potentiation (LTP), has recently given way to new insights into the cellular and molecular mechanisms that underlie acquisition and consolidation of fear memories. Collectively, these findings suggest that fear memory consolidation in the amygdala shares essential biochemical features with LTP, and hold promise for understanding the relationship between memory consolidation and synaptic plasticity in the mammalian brain.     

Hitting Ras where it counts: Ras antagonism in the basolateral amygdala inhibits long-term fear memory

Several studies have implicated the Ras/mitogen-activated protein kinase (MAPK) pathway in Pavlovian fear conditioning. RasGRF1 knockout mice show significant deficits in acquisition of long-term fear memories and long-term potentaition (LTP) in the basolateral amygdala (BLA). MAPK kinase inhibition also impairs fear conditioning and amygdaloid LTP. However, there is no direct evidence to date for the involvement of Ras itself in fear conditioning. To address this issue, we examined the effects of intra-amygdala infusions of the selective Ras antagonist farnesylthiosalicylic acid (FTS) on the acquisition and expression of conditional freezing in rats. Micro-infusions of FTS into the BLA prior to contextual fear conditioning significantly impaired acquisition of long-term contextual fear memory in a dose-dependent manner. Post-training FTS infusions had no effect on acquisition of long-term fear memory. The effects of FTS on fear conditioning were specific for the BLA. Finally, intra-amygdala infusions of FTS inhibited MAPK activation in BLA. Collectively, these results provide further evidence for the involvement of amygdaloid Ras in the acquisition of long-term conditional fear memory.     

L-type voltage-gated calcium channels mediate NMDA-independent associative long-term potentiation at thalamic input synapses to the amygdala.

Long-term potentiation (LTP) in the amygdala is a leading candidate mechanism to explain fear conditioning, a prominent model of emotional memory. LTP occurs in the pathway from the auditory thalamus to the lateral amygdala, and during fear conditioning LTP-like changes occur in the synapses of this pathway. Nevertheless, LTP has not been investigated in the thalamoamygdala pathway using in vitro recordings; hence little is known about the underlying mechanisms. We therefore examined thalamoamygdala LTP in vitro using visualized whole-cell patch recording. LTP at these synapses was dependent on postsynaptic calcium entry, similar to synaptic plasticity in other regions of the brain. However, unlike many forms of synaptic plasticity, thalamoamygdala LTP was independent of NMDA receptors, despite their presence at these synapses, and instead was dependent on L-type voltage-gated calcium channels. This was true when LTP was induced by pairing presynaptic activity with either action potentials or constant depolarization in the postsynaptic cell. In addition, the LTP was associative, in that it required concurrent pre- and postsynaptic activity, and it was synapse specific. Thus, although this LTP is different from that described at other synapses in the brain, it is nonetheless well suited to mediate classical fear conditioning.     

Memory consolidation of Pavlovian fear conditioning requires nitric oxide signaling in the lateral amygdala

Nitric oxide (NO) has been widely implicated in synaptic plasticity and memory formation. In studies of long-term potentiation (LTP), NO is thought to serve as a 'retrograde messenger' that contributes to presynaptic aspects of LTP expression. In this study, we examined the role of NO signaling in Pavlovian fear conditioning. We first show that neuronal nitric oxide synthase is localized in the lateral nucleus of the amygdala (LA), a critical site of plasticity in fear conditioning. We next show that NO signaling is required for LTP at thalamic inputs to the LA and for the long-term consolidation of auditory fear conditioning. Collectively, the findings suggest that NO signaling is an important component of memory formation of auditory fear conditioning, possibly as a retrograde signal that participates in presynaptic aspects of plasticity in the LA.     

Brain-derived neurotrophic factor in amygdala-dependent learning.

The neurotrophin brain-derived neurotrophic factor (BDNF) has recently emerged as a possible molecular mediator of activity-dependent synaptic plasticity underlying learning and memory. Long-term potentiation (LTP) within the hippocampus and hippocampally dependent behaviors has been the primary model for examining the role of BDNF in learning and memory. However, these studies are limited by an incomplete understanding of the complex behavioral function of hippocampal circuitry, making it difficult to unravel the molecular machinery responsible for the formation and storage of these memories. In contrast, the amygdala and its role in Pavlovian fear conditioning promise to provide us with new insights into the mechanisms of BDNF-mediated synaptic plasticity during the learning and memory process. This article reviews the different levels of research on BDNF in learning and memory. The focus is primarily on the use of Pavlovian fear conditioning as a learning model that allows for the examination of the role of BDNF in the amygdala, following a single learning session and within a well-understood neural circuit.     

Long-term potentiation in freely moving rats reveals asymmetries in thalamic and cortical inputs to the lateral amygdala

Long-term memory underlying Pavlovian fear conditioning is believed to involve plasticity at sensory input synapses in the lateral nucleus of the amygdala (LA). A useful physiological model for studying synaptic plasticity is long-term potentiation (LTP). LTP in the LA has been studied only in vitro or in anaesthetized rats. Here, we tested whether LTP can be induced in auditory input pathways to the LA in awake rats, and if so, whether it persists over days. In chronically implanted rats, extracellular field potentials evoked in the LA by stimulation of the auditory thalamus and the auditory association cortex, using test simulations and input/output (I/O) curves, were compared in the same animals after tetanization of either pathway alone or after combined tetanization. For both pathways, LTP was input-specific and long lasting. LTP at cortical inputs exhibited the largest change at early time points (24 h) but faded within 3 days. In contrast, LTP at thalamic inputs, though smaller initially than cortical LTP, remained stable until at least 6 days. Comparisons of I/O curves indicated that the two pathways may rely on different mechanisms for the maintenance of LTP and may benefit differently from their coactivation. This is the first report of LTP at sensory inputs to the LA in awake animals. The results reveal important characteristics of synaptic plasticity in neuronal circuits of fear memory that could not have been revealed with in vitro preparations, and suggest a differential role of thalamic and cortical auditory afferents in long-term memory of fear conditioning.     

Functional neuroanatomy of amygdalohippocampal interconnections and their role in learning and memory

The amygdalar nuclear complex and hippocampal/parahippocampal region are key components of the limbic system that play a critical role in emotional learning and memory. This Review discusses what is currently known about the neuroanatomy and neurotransmitters involved in amygdalo‐hippocampal interconnections, their functional roles in learning and memory, and their involvement in mnemonic dysfunctions associated with neuropsychiatric and neurological diseases. Tract tracing studies have shown that the interconnections between discrete amygdalar nuclei and distinct layers of individual hippocampal/parahippocampal regions are robust and complex. Although it is well established that glutamatergic pyramidal cells in the amygdala and hippocampal region are the major players mediating interconnections between these regions, recent studies suggest that long‐range GABAergic projection neurons are also involved. Whereas neuroanatomical studies indicate that the amygdala only has direct interconnections with the ventral hippocampal region, electrophysiological studies and behavioral studies investigating fear conditioning and extinction, as well as amygdalar modulation of hippocampal‐dependent mnemonic functions, suggest that the amygdala interacts with dorsal hippocampal regions via relays in the parahippocampal cortices. Possible pathways for these indirect interconnections, based on evidence from previous tract tracing studies, are discussed in this Review. Finally, memory disorders associated with dysfunction or damage to the amygdala, hippocampal region, and/or their interconnections are discussed in relation to Alzheimer's disease, posttraumatic stress disorder (PTSD), and temporal lobe epilepsy. © 2016 Wiley Periodicals, Inc.     

Activation of TrkB by 7,8-Dihydroxyflavone Prevents Fear Memory Defects and Facilitates Amygdalar Synaptic Plasticity in Aging

7,8-dihydroxyflavone (7,8-DHF) has recently been identified as a brain-derived neurotrophic factor (BDNF) mimetic to selectively activate the tropomyosin-related kinase B (TrkB) with high affinity. We have previously demonstrated that 7,8-DHF in vitro rescues long-term synaptic plasticity in the hippocampus of aged rats. The present study assessed the effectiveness of 7,8-DHF on age-related declines in fear memories and amygdalar synaptic plasticity. We found that Sprague Dawley male rats began to show significant deficits in the acquisition and retention of memories for contextual and cued fear conditioning, as well as the reduction of BDNF, TrkB, and phosphorylated TrkB at the age of 25 months. Therefore, rats at 24 months old received intraperitoneal administration of either 7,8-DHF (5 mg/kg, i.p.) or vehicle once daily for a consecutive 4 weeks. At the end of treatment period, cognitive performance, amygdalar synaptic plasticity, synaptogenesis, and the phosphorylation of several proteins crucial to synaptic plasticity were evaluated. The results show that chronic 7,8-DHF treatments significantly enhanced the activation of phosphorylated TrkB at the Y515 and Y816 sites, increased spine density and number in several brain regions that process fear memory including the amygdala, hippocampus, and prefrontal cortex, facilitated basolateral amygdalar synaptic plasticity, and in turn prevented performance in fear conditioning tasks from declining. Our results thus confirm a critical role for TrkB signaling activation by 7,8-DHF in preventing age-related declines in fear learning and memory and strongly suggest a potential usefulness for 7,8-DHF or a TrkB agonist in reversing age-related memory impairment.     

Expression of trkB and trkC receptors and their ligands brain-derived neurotrophic factor and neurotrophin-3 in the murine amygdala

The neurotrophin brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) and their cognate receptors, trkB and trkC, have a variety of physiological brain functions, ranging from cell survival to mechanisms involved in learning and memory and long-term potentiation (LTP). LTP can be induced in the cortex and hippocampus, as well as within the amygdala. However, the role of neurotrophins in amygdalar LTP is largely unknown. Expression patterns of BDNF and NT-3 and their cognate receptors in the adult mouse amygdala have not been analyzed in detail. We have therefore examined the expression of trkB, trkC, BDNF, and NT-3 mRNA and protein in different amygdalar nuclei as well as in the hippocampal areas CA1-CA3 and the dentate gyrus. The distribution pattern of trkB, trkC, BDNF, and NT-3 mRNA in the murine hippocampus is comparable to that seen in rats. Within most amygdalar nuclei, a moderate BDNF mRNA expression was found; however, BDNF mRNA was virtually absent from the central nucleus. No expression of NT-3 mRNA was found within the amygdala, but trkC mRNA-expressing cells were widely distributed within this brain region. trkB mRNA was strongly expressed in the amygdala. Because trkB is expressed in a full-length and a truncated form (the latter form is also expressed by nonneuronal cells), we also investigated the distribution of full-length trkB mRNA-expressing cells and could demonstrate that this version of trkB receptors is also widely expressed in the amygdala. These results can serve as a basis for studies elucidating the physiological roles of these receptors in the amygdala.     

Effects of unconditioned and conditioned aversive stimuli in an intense fear conditioning paradigm on synaptic plasticity in the hippocampal CA1 area in vivo

Repeated vivid recalls or flashbacks of traumatic memories and memory deficits are the cardinal features of post-traumatic stress disorder (PTSD). The underlying mechanisms are not fully understood yet. Here, we examined the effects of very strong fear conditioning (20 pairings of a light with a 1.5-mA, 0.5-s foot shock) and subsequent reexposure to the conditioning context (chamber A), a similar context (chamber B), and/or to the fear conditioned stimulus (CS) (a light) on synaptic plasticity in the hippocampal CA1 area in anesthetized Sprague-Dawley rats. The conditioning procedure resulted in very strong conditioned fear, as reflected by high levels of persistent freezing, to both the contexts and to the CS, 24 h after fear conditioning. The induction of long-term potentiation (LTP) was blocked immediately after fear conditioning. It was still markedly impaired 24 h after fear conditioning; reexposure to the conditioning chamber A (CA) or to a similar chamber B (CB) did not affect the impairment. However, presentation of the CS in the CA exacerbated the impairment of LTP, whereas the CS presentation in a CB ameliorated the impairment so that LTP induction did not differ from that of control groups. The induction of long-term depression (LTD) was facilitated immediately, but not 24 h, after fear conditioning. Only reexposure to the CS in the CA, but not reexposure to either chamber A or B alone, or the CS in chamber B, 24 h after conditioning, reinstated the facilitation of LTD induction. These data demonstrate that unconditioned and conditioned aversive stimuli in an intense fear conditioning paradigm can have profound effects on hippocampal synaptic plasticity, which may aid to understand the mechanisms underlying impairments of hippocampus-dependent memory by stress or in PTSD.     

Synaptic transmission changes in fear memory circuits underlie key features of an animal model of schizophrenia

Non-competitive antagonists of the N-methyl-d-aspartate receptor (NMDA) such as phencyclidine (PCP) elicit schizophrenia-like symptoms in healthy individuals. Similarly, PCP dosing in rats produces typical behavioral phenotypes that mimic human schizophrenia symptoms. Although schizophrenic behavioral phenotypes of the PCP model have been extensively studied, the underlying alterations of intrinsic neuronal properties and synaptic transmission in relevant limbic brain microcircuits remain elusive. Acute brain slice electrophysiology and immunostaining of inhibitory neurons were used to identify neuronal circuit alterations of the amygdala and hippocampus associated with changes in extinction of fear learning in rats following PCP treatment. Subchronic PCP application led to impaired long-term potentiation (LTP) and marked increases in the ratio of NMDA to 2-amino-3(5-methyl-3-oxo-1,2-oxazol-4-yl)propionic acid (AMPA) receptor-mediated currents at lateral amygdala (LA) principal neurons without alterations in parvalbumin (PV) as well as non-PV, glutamic acid decarboxylase 67 (GAD 67) immunopositive neurons. In addition, LTP was impaired at the Schaffer collateral to CA1 hippocampal pathway coincident with a reduction in colocalized PV and GAD67 immunopositive neurons in the CA3 hippocampal area. These effects occurred without changes in spontaneous events or intrinsic membrane properties of principal cells in the LA. The impairment of LTP at both amygdalar and hippocampal microcircuits, which play a key role in processing relevant survival information such as fear and extinction memory concurred with a disruption of extinction learning of fear conditioned responses. Our results show that subchronic PCP administration in rats impairs synaptic functioning in the amygdala and hippocampus as well as processing of fear-related memories.     

Aquaporin-4 deficiency impairs synaptic plasticity and associative fear memory in the lateral amygdala: involvement of downregulation of glutamate transporter-1 expression

Astrocytes are implicated in information processing, signal transmission, and regulation of synaptic plasticity. Aquaporin-4 (AQP4) is the major water channel in adult brain and is primarily expressed in astrocytes. A growing body of evidence indicates that AQP4 is a potential molecular target for the regulation of astrocytic function. However, little is known about the role of AQP4 in synaptic plasticity in the amygdala. Therefore, we evaluated long-term potentiation (LTP) in the lateral amygdala (LA) and associative fear memory of AQP4 knockout (KO) and wild-type mice. We found that AQP4 deficiency impaired LTP in the thalamo-LA pathway and associative fear memory. Furthermore, AQP4 deficiency significantly downregulated glutamate transporter-1 (GLT-1) expression and selectively increased NMDA receptor (NMDAR)-mediated EPSCs in the LA. However, low concentration of NMDAR antagonist reversed the impairment of LTP in KO mice. Upregulating GLT-1 expression by chronic treatment with ceftriaxone also reversed the impairment of LTP and fear memory in KO mice. These findings imply a role for AQP4 in synaptic plasticity and associative fear memory in the amygdala by regulating GLT-1 expression.     

Effects of stress and corticosterone on activity and plasticity in the amygdala

The basolateral amygdala (BLA) has been repeatedly shown to mediate the effects of stress on memory-related processes. However, the way in which stress influences BLA itself has not been fully explored. We studied the effects of stress and corticosterone (CORT) on activity and plasticity in the BLA in the rat, using the electrophysiological procedure of long-term potentiation (LTP) induction in vivo. Rats were exposed to an acute elevated-platform stress or administered vehicle or 5 mg/kg, 10 mg/kg, or 25 mg/kg of CORT systemically, after which they were anesthetized and prepared for field potential recording in the BLA, in response to stimulation of the entorhinal cortex. The elevated platform stress enhanced baseline responses in BLA and plasma CORT but inhibited amygdalar LTP. Systemic injections of CORT enhanced baseline responses in BLA in a dose-dependent manner but did not influence amygdalar LTP. Posttetanic potentiation (PTP) was similarly reduced in CORT- and vehicle-injected groups, possibly because of an additional stress from the injection, thus implying that PTP and LTP in the amygdala differentially react to stress. These results suggest that the increase in amygdalar baseline activity following the exposure to stress may be mediated by the concomitant increase in plasma CORT. However, the suppression of amygdalar LTP is not a result of elevated levels of CORT, suggesting that activity and plasticity in the amygdala might be mediated by different mechanisms.     

The striatal-enriched protein tyrosine phosphatase gates long-term potentiation and fear memory in the lateral amygdala.

BACKGROUND: Formation of long-term memories is critically dependent on extracellular-regulated kinase (ERK) signaling. Activation of the ERK pathway by the sequential recruitment of mitogen-activated protein kinases is well understood. In contrast, the proteins that inactivate this pathway are not as well characterized. METHODS: Here we tested the hypothesis that the brain-specific striatal-enriched protein tyrosine phosphatase (STEP) plays a key role in neuroplasticity and fear memory formation by its ability to regulate ERK1/2 activation. RESULTS: STEP co-localizes with the ERKs within neurons of the lateral amygdala. A substrate-trapping STEP protein binds to the ERKs and prevents their nuclear translocation after glutamate stimulation in primary cell cultures. Administration of TAT-STEP into the lateral amygdala (LA) disrupts long-term potentiation (LTP) and selectively disrupts fear memory consolidation. Fear conditioning induces a biphasic activation of ERK1/2 in the LA with an initial activation within 5 minutes of training, a return to baseline levels by 15 minutes, and an increase again at 1 hour. In addition, fear conditioning results in the de novo translation of STEP. Inhibitors of ERK1/2 activation or of protein translation block the synthesis of STEP within the LA after fear conditioning. CONCLUSIONS: Together, these data imply a role for STEP in experience-dependent plasticity and suggest that STEP modulates the activation of ERK1/2 during amygdala-dependent memory formation. The regulation of emotional memory by modulating STEP activity may represent a target for the treatment of psychiatric disorders such as posttraumatic stress disorder (PTSD), panic, and anxiety disorders.     

Effect of mediodorsal thalamic nucleus lesion on contextual fear conditioning in rats

Much evidence from animal and clinical studies has shown that the mediodorsal nucleus of the thalamus (MD) is related to various types of memory, such as visual recognition, object-reward association, spatial working, and reference memory; however, few studies have investigated its role in emotion-related learning and memory processes. This study compared the effect of pre- and posttraining bilateral lesions of the mediodorsal thalamic nucleus with those of the amygdala on contextual conditioned fear. Both pre- and posttraining amygdala lesions almost eliminated conditioned freezing, and significantly blocked postshock freezing when behavioral tests were performed immediately after footshocks, reconfirming previous studies that the amygdala is implicated in the learning of Pavlovian conditioning. Both pre- and posttraining lesions of the mediodorsal nucleus of the thalamus significantly attenuated conditioned freezing but had no effect on postshock freezing. In contrast to lesions of the amygdala, those of the mediodorsal thalamic nucleus failed to alter the increased defecation induced by conditioned fear stress. Our results suggest that the mediodorsal nucleus of the thalamus has an important role in acquisition, consolidation or retrieval in Pavlovian contextual fear conditioning. Possible neural circuits, incorporating the amygdala, MD, and hippocampus, and the functional similarity of the MD and hippocampus in contextual fear conditioning, are also discussed.