Neurotoxic lesions of retrosplenial cortex disrupt signaled and unsignaled contextual fear conditioning

The majority of research regarding contextual learning and memory has focused on the contributions of the hippocampus and related medial temporal lobe structures. However, little is known about other possible cortical contributions to these processes. Our laboratory recently demonstrated that electrolytic lesions of the retrosplenial cortex (RSP), a posterior region of cingulate cortex, impaired contextual but not cue-specific fear conditioning. The present experiments further examined the role of RSP in contextual fear memory using fiber-sparing neurotoxic lesions and both signaled and unsignaled fear conditioning paradigms. Despite comparable acquisition of the conditioned fear response, rats with neurotoxic lesions of RSP exhibited impaired contextual memory relative to control animals in both the signaled and unsignaled paradigms. These results further suggest an important role for RSP in contextual learning and memory.     

Disruption of reconsolidation but not consolidation of auditory fear conditioning by noradrenergic blockade in the amygdala

Consolidation is a process through which labile memories are made persistent [Science 287 (2000) 248]; [Annu Rev Psychol 55 (2004) 51]. When retrieved, a consolidated memory is rendered labile again and undergoes reconsolidation [Learn Mem 7 (2000) 73]; [Trends Neurosci 26 (2003) 65]). Reconsolidation thus offers the opportunity to manipulate memory after it is formed, and may therefore provide a means of treating intrusive memories associated with post-traumatic stress disorder (PTSD). Reconsolidation is most usually studied using protein synthesis inhibitors, which is not practical in humans. However, the beta adrenergic receptor antagonist propranolol impairs consolidation of declarative memory in humans [Science 287 (2000) 248]; [Nature 371 (1994) 702] and consolidation and reconsolidation of inhibitory avoidance learning in rats [Brain Res 368 (1986) 125]; [J Neurosci 19 (1999) 6623]. Here, we show that systemic or intra-amygdala infused propranolol blocks reconsolidation but not consolidation. If the effects on reconsolidation are verified in humans, the results would suggest the possibility that propranolol after memory retrieval might be an effective way of treatment of intrusive memories in PTSD. That the systemic effects of propranolol on reconsolidation are achieved via an action in the amygdala is especially important in light of the fact that PTSD involves alterations in the amygdala [Arch Gen Psychiatry 53 (1996) 380].     

The formation of auditory fear memory requires the synthesis of protein and mRNA in the auditory thalamus

The medial geniculate nucleus of the thalamus responds to auditory information and is a critical part of the neural circuitry underlying aversive conditioning with auditory signals for shock. Prior work has shown that lesions of this brain area selectively disrupt conditioning with auditory stimuli and that neurons in the medial geniculate demonstrate plastic changes during fear conditioning. However, recent evidence is less clear as to whether or not this area plays a role in the storage of auditory fear memories. In the current set of experiments rats were given infusions of protein or messenger RNA (mRNA) synthesis inhibitors into the medial geniculate nucleus of the thalamus 30 min prior to auditory fear conditioning. The next day animals were tested to the auditory cue and conditioning context. Results showed that rats infused with either inhibitor demonstrated less freezing to the auditory cue 24 h after training, while freezing to the context was normal. Autoradiography confirmed that the doses used were effective in disrupting synthesis. Taken together with prior work, these data suggest that the formation of fear memory requires the synthesis of new protein and mRNA at multiple brain sites across the neural circuit that supports fear conditioning.     

Low-probability transmission of neocortical and entorhinal impulses through the perirhinal cortex

One model of episodic memory posits that during slow-wave sleep (SWS), the synchronized discharges of hippocampal neurons in relation to sharp waves "replay" activity patterns that occurred during the waking state, facilitating synaptic plasticity in the neocortex. Although evidence of replay was found in the hippocampus in relation to sharp waves, it was never shown that this activity reached the neocortex. Instead, it was assumed that the rhinal cortices faithfully transmit information from the hippocampus to the neocortex and reciprocally. Here, we tested this idea using 3 different approaches. 1) Stimulating electrodes were inserted in the entorhinal cortex and temporal neocortex and evoked unit responses were recorded in between them, in the intervening rhinal cortices. In these conditions, impulse transfer occurred with an extremely low probability, in both directions. 2) To rule out the possibility that this unreliable transmission resulted from the artificial nature of electrical stimuli, crosscorrelation analyses of spontaneous neocortical, perirhinal, and entorhinal firing were performed in unanesthetized animals during the states of waking and SWS. Again, little evidence of propagation could be obtained in either state. 3) To test the idea that propagation occurs only when large groups of neurons are activated within a narrow time window, we computed perievent histograms of neocortical, perirhinal, and entorhinal neuronal discharges around large-amplitude sharp waves. However, these synchronized entorhinal discharges also failed to propagate across the perirhinal cortex. These findings suggest that the rhinal cortices are more than a relay between the neocortex and hippocampus, but rather a gate whose properties remain to be identified.     

Patterns of Fos expression in the amygdala and ventral perirhinal cortex induced by training in an olfactory fear conditioning paradigm

The activation of amygdaloid nuclei, the ventral perirhinal cortex (vPRh), and several other brain areas in the rat during the acquisition and expression of olfactory fear conditioning was assessed through Fos immunocytochemistry in 3 separate experiments. The results of Experiment 1 suggest that olfactory and somatosensory inputs may functionally converge in the anterior region ot the medial nucleus (aMe). The results of Experiment 2 indicate that the aMe exhibited significantly greater Fos-like immunoreactivity (FLI) in subjects acquiring conditioned stimulus-unconditioned stimulus associations than in those presented with the same olfactory and somatosensory stimuli in a manner that precluded acquisition. The results of Experiment 3 indicate that the vPRh appeared to exhibit learning-related increases in FLI during the expression of previously acquired associations. Collectively, these data suggest that the aMe and vPRh may be critically involved in different aspects of olfactory fear conditioning.     

N-methyl-D-aspartate receptor antagonist APV blocks acquisition but not expression of fear conditioning.

The role of N-methyl-D-aspartate (NMDA) receptors in Pavlovian fear conditioning was examined using the NMDA antagonist DL-2-amino-5-phosphonovaleric acid (APV). Either APV (5 micrograms/rat) or saline was administered before the training phase, the testing phase, or both. APV completely blocked acquisition but not expression of fear conditioning. The L enantiomer of APV did not affect the acquisition of conditional fear. To separate encoding from consolidation processes, APV was administered either before or immediately after the footshock unconditional stimulus (US) during the training phase. The results indicate that APV must be present during the US to produce its effects on fear conditioning. The behavioral effect of the drug is not due to analgesic action because APV did not alter pain sensitivity. The data suggest that NMDA receptors are critical for the acquisition but not expression of fear conditioning. These effects on fear conditioning are parallel to the in vitro effects of APV on the acquisition but not expression of long-term potentiation (LTP) and suggest that endogenously generated NMDA-dependent LTP participates in the neural plasticity underlying fear conditioning.     

Anterograde tracer and field potential analysis of the neocortical layer I projection from nucleus ventralis medialis of the thalamus in cat

The projection of the ventromedial nucleus of the thalamus to the neocortex was studied in cat by means of anterograde and retrograde transport of horseradish peroxidase, by the depth profile of evoked thalamocortical field potentials, and by superfusion of the cortex with manganese to block transmitter release. Horseradish peroxidase injected into the ventromedial nucleus was anterogradely transported to the outer third of layer I in the neocortex, extending from the depth of the cruciate sulcus anterior to the olfactory bulb and tract. The region of projection from the ventromedial nucleus extended mediolaterally from the medial wall of the proreus gyrus to the ventral tip of the coronal gyrus. Horseradish peroxidase injections or applications in these areas of the neocortex resulted in the retrograde labeling of neurons in the ventromedial nucleus. Injections in many other cortical areas did not result in labeled neurons in this nucleus. Stimulation of the ventromedial nucleus with single pulses elicited surface-negative waves in the medial part of the precruciate region that had superficial isoelectric points. Superfusion of the precruciate area with manganese resulted in the suppression of the ventromedial-evoked wave, whereas control extracellular waves in deeper layers were unaffected. An additional additional finding was that horseradish peroxidase injections in the ventromedial nucleus led to a dense reciprocal retrograde labeling of neurons in layer VI of that part of the cortex to which the ventromedial nucleus projects. We conclude that, in cat, (1) the ventromedial nucleus projects to layer I of the cerebral cortex anterior to the cruciate sulcus and receives a dense reciprocal projection from layer VI; (2) stimulation of neurons in the ventromedial nucleus causes depolarization of structures in layer I and these neurons are responsible for recruiting responses in the anterior cortex.     

Anisomycin does not disrupt the activation of penile reflexes by testosterone in rats

A possible role of protein synthetic processes in the testosterone-activation of penile reflexes in rats was examined in these experiments. In Experiment 1, long-term castrated male rats were injected with 250 micrograms testosterone propionate (TP) and tested for penile reflexes 24 hr later. Fifteen minutes prior to TP these males received a systemic injection of the protein synthesis inhibitor anisomycin (ANI) or the saline vehicle. ANI had no disruptive effect on the activation of penile reflexes by TP; in fact, ANI facilitated erection frequency. In Experiments 2 and 3, a series of three ANI or saline injections were given at 2 hr intervals beginning with the injection of 250 micrograms TP, with no significant effect on any reflex parameters tested 12 or 24 hr after TP. In Experiment 4, the penile reflexes of male rats were stimulated by implanting a Silastic capsule containing testosterone subcutaneously for 2 weeks. A series of ANI or saline injections were spaced 3 hr apart, with penile reflexes tested 6 and 12 hr after the first injection. There were no significant differences between ANI and saline-treated males at 6 hr, whereas at 12 hr ANI-treated males had significantly shorter reflex latencies and significantly more penile flips than did males injected with saline. In a final experiment (Experiment 5), the Silastic capsules were removed from the males in the previous experiment. Three injections of ANI or saline were given at 4 hr intervals beginning with the removal of the Silastic capsule.(ABSTRACT TRUNCATED AT 250 WORDS)     

Development of reorganization of the auditory cortex caused by fear conditioning: effect of atropine

Reorganization of the frequency map in the central auditory system is based on shifts in the best frequencies (BFs; hereafter, BF shifts), together with the frequency-response curves, of auditory neurons. In the big brown bat, conditioning with acoustic stimulation followed by electric leg-stimulation causes BF shifts of collicular and cortical neurons. The collicular BF shift develops quickly and is short term, whereas the cortical BF shift develops slowly and is long term. The acetycholine level in the auditory cortex must be high during conditioning to develop these BF shifts. We studied the effect of atropine (an antagonist of muscarinic acetylcholine receptors) applied to the auditory cortex on the development of the long-term cortical BF shift in the awake bat caused by a 30-min conditioning session. We found 1) the cortical BF shift starts to develop approximately 15 min after the onset of the conditioning, gradually increases over 60 min, and reaches a plateau, 2) the cortical BF shift changes from short to long term approximately 45 min after the onset of the conditioning, 3) the cortical BF shift can plateau at different frequencies between the BF of a given neuron in the control condition and the frequency of the conditioning tone, 4) the maximum BF shift is determined approximately 70 min after the onset of the conditioning, and 5) acetylcholine plays an important role in the development of the cortical BF shift. Its role ends approximately 180 min after the onset of the conditioning.     

D-baclofen does not antagonize the actions of L-baclofen on rat neocortical neurons in vitro

The ability of D-baclofen to antagonize the actions of L-baclofen on rat neocortical neurons was investigated. Intracellular recordings were made from neurons in cortical layers 2 and 3 in an in vitro slice preparation. Baclofen stereoisomers were applied at known concentrations in the superfusion medium. At a concentration of 3 microM, L-baclofen produced approximately 70% depressions of excitatory and inhibitory postsynaptic potentials (EPSPs and IPSPs) that were evoked by stimulation of superficial cortical layers. L-baclofen also hyperpolarized neocortical neurons. These hyperpolarizations were accompanied by decreases in neuronal input resistance and in direct excitability. We have shown previously that these latter effects are secondary to the action of baclofen to increase the potassium conductance of neocortical neurons. D-baclofen, at concentrations of 1-100 microM, did not antagonize depressions by L-baclofen of EPSPs and IPSPs nor the action of L-baclofen to increase the potassium conductance of neocortical neurons. At concentrations of 50-100 microM, D-baclofen produced 20-30% effects when applied alone, thus suggesting that these concentrations of D-baclofen produced a significant degree of receptor occupancy. Our results demonstrate that D-baclofen is not an antagonist or high affinity partial agonist at the receptors through which baclofen exerts its effects on single neurons in the rat neocortex.     

Azimuthal processing in the posterior auditory thalamus of cats.

The responses to free-field acoustic stimuli of 157 units in the auditory thalamus of anesthetized cats were studied in relation to the localization of pure tone stimuli in the azimuthal plane. Units were classified as 'directional' if their firing rates at sound levels in excess of 20 dB above threshold varied by more than 50% as a function of azimuth. Sixty-five % of the units in the nucleus of the brachium of the inferior colliculus and 30% in the ventral division of the medial geniculate body were found to be directional, suggesting different processing channels for sound localization between colliculus and cortex.     

Spectrotemporal receptive fields in the lemniscal auditory thalamus and cortex.

Receptive fields have been characterized independently in the lemniscal auditory thalamus and cortex, usually with spectrotemporally simple sounds tailored to a specific task. No studies have employed naturalistic stimuli to investigate the thalamocortical transformation in temporal, spectral, and aural domains simultaneously and under identical conditions. We recorded simultaneously in the ventral division of the medial geniculate body (MGBv) and in primary auditory cortex (AI) of the ketamine-anesthetized cat. Spectrotemporal receptive fields (STRFs) of single units (n = 387) were derived by reverse-correlation with a broadband and dynamically varying stimulus, the dynamic ripple. Spectral integration, as measured by excitatory bandwidth and spectral modulation preference, was similar across both stations (mean Q(1/e) thalamus = 5.8, cortex = 5.4; upper cutoff of spectral modulation transfer function, thalamus = 1.30 cycles/octave, cortex = 1.37 cycles/octave). Temporal modulation rates slowed by a factor of two from thalamus to cortex (mean preferred rate, thalamus = 32.4 Hz, cortex = 16.6 Hz; upper cutoff of temporal modulation transfer function, thalamus = 62.9 Hz, cortex = 37.4 Hz). We found no correlation between spectral and temporal integration properties, suggesting that the excitatory-inhibitory interactions underlying preference in each domain are largely independent. A small number of neurons in each station had highly asymmetric STRFs, evidence of frequency sweep selectivity, but the population showed no directional bias. Binaural preferences differed in their relative proportions, most notably an increased prevalence of excitatory contralateral-only cells in cortex (40%) versus thalamus (23%), indicating a reorganization of this parameter. By comparing simultaneously along multiple stimulus dimensions in both stations, these observations establish the global characteristics of the thalamocortical receptive field transformation.     

Network activity evoked by neocortical stimulation in area 36 of the guinea pig perirhinal cortex.

The perirhinal cortex is a key structure involved in memory consolidation and retrieval. In spite of the extensive anatomical studies that describe the intrinsic and extrinsic associative connections of the perirhinal cortex, the activity generated within such a network has been poorly investigated. We describe here the pattern of synaptic interactions that subtend the responses evoked in area 36 of the perirhinal cortex by neocortical and local stimulation. The experiments were carried out in the in vitro isolated guinea pig brain. The synaptic perirhinal circuit was reconstructed by integrating results obtained during intracellular recordings from layer II-III neurons with simultaneous current source density analysis of laminar profiles performed with 16-channel silicon probes. Both neocortical and local stimulation of area 36 determined a brief monosynaptic excitatory potential in layer II-III neurons, followed by a biphasic synaptic inhibitory potential possibly mediated by a feed-forward inhibitory circuit at sites close to the stimulation electrode and a late excitatory postsynaptic potential (EPSP) that propagated at distance within area 36 along the rhinal sulcus. During a paired-pulse stimulation test, the inhibitory postsynaptic potential (IPSP) and the late EPSP were abolished in the second conditioned response, suggesting that they are generated by poli-synaptic circuits. Current source density analysis of the field responses demonstrated that 1) the monosynaptic activity was generated in layers II-III and 2) the sink associated to the disynaptic responses was localized within the superficial layer of area 36. We conclude that the neocortical input induces a brief monosynaptic excitation in area 36 of the perirhinal cortex, that is curtailed by a prominent inhibition and generates a recurrent excitatory associative response that travels at distance within area 36 itself. The results suggest that the perirhinal cortex network has the potentials to integrate multimodal incoming neocortical information on its way to the hippocampus.     

Pretraining tetanic fimbrial stimulation impairs the expression but not the acquisition of contextual fear conditioning in mice.

We recently reported that the pretraining induction of long-term potentiation in the lateral septum by fimbrial tetanic stimulation altered contextual fear conditioning in mice. The aim of the present study was to examine at which stage of fear conditioning (i.e. either acquisition or expression) this impairment takes place. Mice implanted with stimulating electrodes in the fimbria and recording electrodes in the lateral septal were conditioned to acquire fear towards a novel context using a footshock procedure. Twenty-four hours after conditioning, animals were re-exposed to the conditioning environment and the level of freezing behavior served as the measure of conditioned fear. The level of fimbrial-lateral septal synaptic neurotransmission was manipulated using either fimbrial tetanic stimulation (which induced septal long-term potentiation) alone, or followed by fimbrial low-frequency stimulation producing depotentiation of the previously established long-term potentiation. The results showed that (i) septal long-term potentiation induced either prior to acquisition or only prior to retention testing impaired conditioned freezing; and (ii) the impairing effect of pretraining induction of long-term potentiation on conditioned freezing was not only abolished by fimbrial low-frequency stimulation administered prior to retention testing but actually produced enhanced conditioned freezing with respect to controls. These data suggest that the level of fimbrial-lateral septal synaptic neurotransmission may influence the expression, but not the acquisition, of contextual fear conditioning.     

GABAergic regulation of auditory sensory gating in low- and high-anxiety rats submitted to a fear conditioning procedure

The inferior colliculus (IC) is primarily involved in the processing of acoustic stimuli, being in a position to send auditory information to motor centers that participate in behaviors such as prey catching and predators' avoidance. The role of the central nucleus of the IC (CIC) on fear and anxiety has been suggested on the basis that rats are able to engage in tasks to decrease the aversiveness of CIC stimulation, increased Fos immunolabeling during diverse aversive states and increased CIC auditory evoked potentials (AEP) induced by conditioned fear stimuli. Additionally, it was shown that brainstem AEP, represented by wave V, for which the main generator is the IC, is increased during experimentally-induced anxiety. Rats segregated according to their low or high emotional reactivity have been used as an important tool in the study of fear and anxiety. The IC contains a high density of GABA receptors. Since the efficacy of an anxiolytic compound is a function of the animal's anxiety level, it is possible that GABA-benzodiazepine (Bzp) agents affect LA and HA animals differently. In this study we investigated the GABA-Bzp influence on the modulation of AEP in rats with low- (LA) or high-anxiety (HA) levels, as assessed by the elevated plus-maze test (EPM). GABA-Bzp modulation on the unconditioned AEP response was analyzed by using intra-CIC injections (0.2 μl) of the GABA-Bzp agonists muscimol (121 ng) and diazepam (30 μg), or the GABA inhibitors bicuculline (10 ng) and semicarbazide (7 μg). In a second experiment, we evaluate the effects of contextual aversive conditioning on AEP using foot-shocks as unconditioned stimuli. On the unconditioned fear paradigm GABA inhibition increased AEP in LA rats and decreases this measure in HA counterparts. Muscimol was effective in reducing AEP in both LA and HA rats. Contextual fear stimuli increased the magnitude of AEP. In spite of no effect obtained with diazepam in LA rats the drug inhibited AEP in HA animals. The specificity of the regulatory mechanisms mediated by GABA-Bzp for the ascending neurocircuits responsible for the acquisition of aversive information in LA and HA animals shed light on the processing of sensory information underlying the generation of defensive reactions.     

Differential impairment of auditory and contextual fear conditioning by protein synthesis inhibition in C57BL/6N mice.

A 1-trial fear conditioning was used to investigate the temporal development of fear responses expressed as increase of freezing or heart rate and its impairment by the protein synthesis inhibitor cycloheximide (CHX) in male C57BL/6N mice. Heart rate was measured with an implanted transmitter. In the memory tests, mice were exposed to tone and context provided either as foreground or background stimulus during training. The fear responses developed differently from 0 to 24 hr after training under these 3 conditions. A single pretraining CHX injection impaired both memory forms, whereas a single posttraining CHX injection impaired tone- but not context-dependent memory, with the context provided as background stimulus. It was concluded that consolidation of tone-, foreground context-, and background context-dependent fear conditioning may be mediated by partly different neuronal or partly different biochemical pathways, or both.     

Effect of lesions in the lateral nucleus of the amygdala on fear conditioning using auditory and visual conditioned stimuli in rats

The lateral nucleus of the amygdala (LA) is believed to be the site of auditory conditioned stimulus (CS) relay in classical fear conditioning. The present study attempts to determine whether the LA is specifically involved in fear conditioning using an auditory CS. Seven rats with lesions in the LA (Tone-Lateral group) and eight sham-operated rats in the control group were trained using an auditory CS (overtone based on an 800 Hz fundamental tone, 70 dB, 3.7 s) paired with foot shock (1.0 mA, 0.5 s). Five rats with lesions in the LA (Light-Lateral group) and eight unoperated rats in the control group were trained using a visual CS (25 W light, 3.7 s). The behavioral index of fear conditioning was a potentiation of the startle reflex in the presence of CS. All rats in the control group and Light-Lateral group showed this potentiation, whereas those in the Tone-Lateral group did not. These results suggest that the LA is an input site of auditory CS information into the amygdala, and that it is not a site of visual CS information input in fear conditioning. Thus, each modality of CS may have a specific subnucleus of the amygdala that mediates fear conditioning.     

Acquisition and generalization of fear conditioning are not modulated by the BDNF-val66met polymorphism in humans

Few studies have investigated the role of the BDNF‐val66met polymorphism in fear conditioning in humans, and previous results have been inconsistent. In the present study, we examined whether the BDNF‐val66met was associated with differences in the acquisition and generalization of fear during a differential conditioning paradigm in a large sample of participants (N = 141). Using three different indexes of fear learning (fear‐potentiated startle, skin conductance response, and online risk ratings) no effects of the BDNF‐val66met were found either on the acquisition or the generalization of conditioned fear. Taken together with previous data, our study suggests that the BDNF‐val66met polymorphism has no effect on the acquisition or generalization of fear.     

Neurons of the acoustic thalamus that project to the amygdala contain glutamate.

Injection of WGA-HRP into the lateral nucleus of the amygdala produced retrograde axonal transport to cell bodies in areas of the acoustic thalamus: the medial division of the medial geniculate body, the suprageniculate nucleus, and the posterior intralaminar nucleus. Glutamate-immunoreactive neurons were present throughout the acoustic thalamus, including the regions containing the retrogradely labeled neurons. Many of the retrogradely labeled cells were also immunoreactive for glutamate. Thus, glutamate is present in those neurons of the acoustic thalamus that project to the amygdala and may contribute to neurotransmission and synaptic plasticity in this pathway.     

Hippocampus and Pavlovian fear conditioning in rats: muscimol infusions into the ventral, but not dorsal, hippocampus impair the acquisition of conditional freezing to an auditory conditional stimulus

The authors compared the effects of pharmacological inactivation of the dorsal hippocampus (DH) or ventral hippocampus (VH) on Pavlovian fear conditioning in rats. Freezing behavior served as the measure of fear. Pretraining infusions of muscimol, a GABAA receptor agonist, into the VH disrupted auditory, but not contextual, fear conditioning; DH infusions did not affect fear conditioning. Pretesting inactivation of the VH or DH did not affect the expression of conditional freezing. Pretraining electrolytic lesions of the VH reproduced the effects of muscimol infusions, whereas posttraining VH lesions disrupted both auditory and contextual freezing. Hence, neurons in the VH are importantly involved in the acquisition of auditory fear conditioning and the expression of auditory and contextual fear under some conditions.