Revisiting the autoconditioning hypothesis for acquired reactivity to ultrasonic alarm calls.

Rats emit 22 kHz ultrasonic vocalizations (USVs) in association with pain, fear, or distress. Whereas the capacity to produce USVs is innate, reactivity to them appears to require experience. Specifically, 22 kHz USVs fail to elicit freezing behavior in naïve laboratory rats. However, these "alarm calls" do elicit freezing in rats that previously experienced foot shocks. These findings led to the hypothesis that acquired reactivity is based on "autoconditioning"-learning in which self-generated 22 kHz USVs serve as Pavlovian cues that become associated with foot shocks. The current study tested the autoconditioning hypothesis by devocalizing rats through a unilateral transection of the recurrent laryngeal nerve (Experimental group). Subsequently, animals in both the Experimental and sham-operated Control groups received five unsignaled foot shocks. One or two days later, both groups were tested for USV-elicited freezing in a novel context. Recurrent laryngeal nerve transection failed to prevent or even diminish USV-elicited freezing. In fact, both groups showed large and comparable increases in freezing to USV presentations. A subset of Control animals failed to vocalize during conditioning, while some Experimental animals did vocalize during conditioning. Animals were therefore re-grouped and reanalyzed based on whether they vocalized during conditioning. Again, both groups showed large and comparable increases in USV-elicited freezing. These results disconfirm the essential tenet or prediction of the autoconditioning hypothesis. Alternative mechanisms for acquired reactivity to 22 kHz USVs are therefore considered.

Muscarinic receptors in amygdala control trace fear conditioning.

Intelligent behavior requires transient memory, which entails the ability to retain information over short time periods. A newly-emerging hypothesis posits that endogenous persistent firing (EPF) is the neurophysiological foundation for aspects or types of transient memory. EPF is enabled by the activation of muscarinic acetylcholine receptors (mAChRs) and is triggered by suprathreshold stimulation. EPF occurs in several brain regions, including the lateral amygdala (LA). The present study examined the role of amygdalar mAChRs in trace fear conditioning, a paradigm that requires transient memory. If mAChR-dependent EPF selectively supports transient memory, then blocking amygdalar mAChRs should impair trace conditioning, while sparing delay and context conditioning, which presumably do not rely upon transient memory. To test the EPF hypothesis, LA was bilaterally infused, prior to trace or delay conditioning, with either a mAChR antagonist (scopolamine) or saline. Computerized video analysis quantified the amount of freezing elicited by the cue and by the training context. Scopolamine infusion profoundly reduced freezing in the trace conditioning group but had no significant effect on delay or context conditioning. This pattern of results was uniquely anticipated by the EPF hypothesis. The present findings are discussed in terms of a systems-level theory of how EPF in LA and several other brain regions might help support trace fear conditioning.

Dual functions of perirhinal cortex in fear conditioning.

The present review examines the role of perirhinal cortex (PRC) in Pavlovian fear conditioning. The focus is on rats, partly because so much is known, behaviorally and neurobiologically, about fear conditioning in these animals. In addition, the neuroanatomy and neurophysiology of rat PRC have been described in considerable detail at the cellular and systems levels. The evidence suggests that PRC can serve at least two types of mnemonic functions in Pavlovian fear conditioning. The first function, termed "stimulus unitization," refers to the ability to treat two or more separate items or stimulus elements as a single entity. Supporting evidence for this perceptual function comes from studies of context conditioning as well as delay conditioning to discontinuous auditory cues. In a delay paradigm, the conditional stimulus (CS) and unconditional stimulus (US) overlap temporally and co-terminate. The second PRC function entails a type of "transient memory." Supporting evidence comes from studies of trace cue conditioning, where there is a temporal gap or trace interval between the CS offset and the US onset. For learning to occur, there must be a transient CS representation during the trace interval. We advance a novel neurophysiological mechanism for this transient representation. These two hypothesized functions of PRC are consistent with inferences based on non-aversive forms of learning.

Rats learn to freeze to 22-kHz ultrasonic vocalizations through autoconditioning.

Rats emit ultrasonic vocalizations (USVs) at ∼22kHz and ∼50kHz, respectively, during negative and positive affective states. Among rats raised in a naturalistic social context, 22-kHz USVs serve as "alarm cries" that can elicit freezing behavior. By contrast, several studies show that naïve laboratory rats do not freeze in response to alarm cries. An obvious and consistent interpretation of these facts is that USV-elicited freezing depends on a type of social learning that ordinarily does not occur in the laboratory. However, the present study explored an alternative and explicitly non-social learning mechanism. Animals in the experimental group received multiple footshocks that elicited 22-kHz USVs. Animals in the control group were exposed to the same chamber but did not receive footshocks and, therefore, did not vocalize. When subsequently tested in a novel context, experimental animals froze in response to a novel 22-kHz USV but were unresponsive to a novel 50-kHz USV. Vocalizing during the aversive experience was predictive of subsequent freezing to the 22-kHz USV. As expected from previous studies, control animals failed to freeze to either USV. We propose that the experimental animals learned to associate their own 22-kHz USVs with an internal fear state and selectively generalized this "autoconditioning" to a novel 22-kHz USV. This non-social form of learning seems sufficiently rapid, reliable, and stimulus-specific to be ethologically adaptive.

Muscarinic receptor activation enables persistent firing in pyramidal neurons from superficial layers of dorsal perirhinal cortex.

Persistent-firing neurons in the entorhinal cortex (EC) and the lateral nucleus of the amygdala (LA) continue to discharge long after the termination of the original, spike-initiating current. An emerging theory proposes that endogenous persistent firing helps support a transient memory system. This study demonstrated that persistent-firing neurons are also prevalent in rat perirhinal cortex (PR), which lies immediately adjacent to and is reciprocally connected with EC and LA. Several characteristics of persistent-firing neurons in PR were similar to those previously reported in LA and EC. Persistent firing in PR was enabled by the application of carbachol, a nonselective cholinergic agonist, and it was induced by injecting a suprathreshold current or by stimulating suprathreshold excitatory synaptic inputs to the neuron. Once induced, persistent firing lasted for seconds to minutes. Persistent firing could always be terminated by a sufficiently large and prolonged hyperpolarizing current; it was prevented by antagonists of muscarinic cholinergic receptors (mAChRs); and it was blocked by flufenamic acid. The latter has been suggested to inhibit a Ca(2+) -activated nonspecific cation conductance (G(CAN) ) that normally furnishes the sustained depolarization during persistent firing. In many PR neurons, the discharge rate during persistent firing was a graded function of depolarizing and/or hyperpolarizing inputs. Persistent firing was not prevented by blocking fast excitatory and inhibitory synaptic transmission, demonstrating that it can be generated endogenously. We suggest that persistent-firing neurons in PR, EC, LA, and certain other brain regions may cooperate in support of a transient-memory system.

Positive and negative ultrasonic social signals elicit opposing firing patterns in rat amygdala.

Rat ultrasonic vocalizations (USVs) are ethologically-essential social signals. Under natural conditions, 22kHz USVs and 50kHz USVs are emitted in association with negative and positive emotional states, respectively. Our first experiment examined freezing behavior elicited in naïve Sprague-Dawley rats by a 22kHz USV, a 50kHz USV, and frequency-matched tones. None of the stimuli elicited freezing, which is the most commonly-used index of fear. The second experiment examined single-unit responses to these stimuli in the amygdala (AM), which is well-known for its role in innate and acquired fear responses. Among 127 well-discriminated single units, 82% were auditory-responsive. Elicited firing patterns were classified using a multi-dimensional scheme that included transient (phasic) responses to the stimulus onsets and/or offsets as well as sustained (tonic) responses during the stimulus. Tonic responses, which are not ordinarily evaluated in AM, were 4.4-times more common than phasic responses. The 22kHz stimuli tended to elicit tonic increases in the firing rates, whereas the 50kHz stimuli more often elicited tonic decreases in firing rates. These opposing tonic responses correspond with the ethological valence of USVs in the two frequency bands. Thus, a relatively-small sample of single-unit responses in AM furnished a more sensitive index of emotional valence than freezing behavior. Latency analysis suggested that stimuli in the two frequency bands are processed through different pathways to AM. One possible interpretation is that phasic responses in AM reflect the detection of a stimulus change, whereas tonic responses indicate the valence of the detected stimulus.

Aging-related changes in calcium-binding proteins in rat perirhinal cortex.

Dysregulation of intracellular calcium homeostasis has been linked to neuropathological symptoms observed in aging and age-related disease. Alterations in the distribution and relative frequency of calcium-binding proteins (CaBPs), which are important in regulating intracellular calcium levels, may contribute to disruption of calcium homeostasis. Here we examined the laminar distribution of three CaBPs in rat perirhinal cortex (PR) as a function of aging. Calbindin-D28k (CB), parvalbumin (PV), and calretinin (CR) were compared in adult (4 mo.), middle-aged (13 mo.) and aged (26 mo.) rats. Results show an aging-related and layer-specific decrease in the number of CB-immunoreactive (-ir) neurons, beginning in middle-aged animals. Dual labeling suggests that the age-related decrease in CB reflects a decrease in neurons that are not immunoreactive for the inhibitory neurotransmitter GABA. In contrast, no aging-related differences in PV- or CR-immunoreactivity were observed. These data suggest that selective alterations in CB-ir neurons may contribute to aging-related learning and memory deficits in tasks that depend upon PR circuitry.

Muscarinic receptors in perirhinal cortex control trace conditioning.

Trace conditioning requires that a transient representation of the conditional stimulus (CS) persists during the time interval between the CS offset and the onset of the unconditional stimulus. According to one hypothesis, this transient CS representation is supported by endogenous activity in "persistent-firing" neurons of perirhinal cortex (PR). By definition, persistent-firing neurons discharge for tens of seconds or minutes after the termination of the original spike-initiating stimulus. This continued spiking does not depend on recurrent circuit activity and can be reliably and completely blocked by muscarinic receptor antagonists. The present study evaluated the role of PR muscarinic receptors in trace fear conditioning. Before conditioning, rats received bilateral intra-PR infusions with either saline or scopolamine, a nonselective muscarinic receptor antagonist. Scopolamine infusions profoundly impaired trace conditioning but had no effect on delay conditioning or context conditioning. The results encourage a more general understanding of muscarinic receptors in PR and they motivate additional tests of the emerging theory that persistent-firing neurons support aspects of transient memory.

Perirhinal cortex supports acquired fear of auditory objects.

Damage to rat perirhinal cortex (PR) profoundly impairs fear conditioning to 22kHz ultrasonic vocalizations (USVs), but has no effect on fear conditioning to continuous tones. The most obvious difference between these two sounds is that continuous tones have no internal temporal structure, whereas USVs consist of strings of discrete calls separated by temporal discontinuities. PR was hypothesized to support the fusion or integration of discontinuous auditory segments into unitary representations or "auditory objects". This transform was suggested to be necessary for normal fear conditioning to occur. These ideas naturally assume that the effect of PR damage on auditory fear conditioning is not peculiar to 22kHz USVs. The present study directly tested these ideas by using a different set of continuous and discontinuous auditory cues. Control and PR-damaged rats were fear conditioned to a 53kHz USV, a 53kHz continuous tone, or a 53kHz discontinuous tone. The continuous and discontinuous tones matched the 53kHz USV in terms of duration, loudness, and principle frequency. The on/off pattern of the discontinuous tone matched the pattern of the individual calls of the 53kHz USV. The on/off pattern of the 50kHz USV was very different from the patterns in the 22kHz USVs that have been comparably examined. Rats with PR damage were profoundly impaired in fear conditioning to both discontinuous cues, but they were unimpaired in conditioning to the continuous cue. The implications of this temporal discontinuity effect are explored in terms of contemporary ideas about PR function.

Imaging the spread of reversible brain inactivations using fluorescent muscimol.

Muscimol is a GABA A-agonist that causes rapid and reversible suppression of neurophysiological activity. Interpretations of the effects of muscimol infusions into the brain have been limited because of uncertainty about spread of the drug around the injection site. To solve this problem, the present study explored the use of a fluorophore-conjugated muscimol molecule (FCM). Whole-cell recordings from horizontal brain slices demonstrated that bath-applied FCM acts like muscimol in reversibly suppressing excitatory synaptic transmission. Two types of in vivo experiments demonstrated that the behavioral effects of FCM infusion are similar to the behavioral effects of muscimol infusion. FCM infusion into the rat amygdala before fear conditioning impaired both cued and contextual freezing, which were tested 24 or 48 h later. Normal fear conditioning occurred when these same rats were subsequently given phosphate-buffered saline infusions. FCM infusion into the dorsomedial prefrontal cortex impaired accuracy during a delayed-response task. Histological analysis showed that the region of fluorescence was restricted to 0.5-1mm from the injection site. Myelinated fiber tracts acted as diffusional barriers, thereby shaping the overall spread of fluorescence. The results suggest that FCM is indeed useful for exploring the function of small brain regions.

Asymmetrical stimulus generalization following differential fear conditioning.

Rodent ultrasonic vocalizations (USVs) are ethologically critical social signals. Rats emit 22kHz USVs and 50kHz USVs, respectively, in conjunction with negative and positive affective states. Little is known about what controls emotional reactivity to these social signals. Using male Sprague-Dawley rats, we examined unconditional and conditional freezing behavior in response to the following auditory stimuli: three 22kHz USVs, a discontinuous tone whose frequency and on-off pattern matched one of the USVs, a continuous tone with the same or lower frequencies, a 4kHz discontinuous tone with an on-off pattern matched to one of the USVs, and a 50kHz USV. There were no differences among these stimuli in terms of the unconditional elicitation of freezing behavior. Thus, the stimuli were equally neutral before conditioning. During differential fear conditioning, one of these stimuli (the CS(+)) always co-terminated with a footshock unconditional stimulus (US) and another stimulus (the CS(-)) was explicitly unpaired with the US. There were no significant differences among these cues in CS(+)-elicited freezing behavior. Thus, the stimuli were equally salient or effective as cues in supporting fear conditioning. When the CS(+) was a 22kHz USV or a similar stimulus, rats discriminated based on the principal frequency and/or the temporal pattern of the stimulus. However, when these same stimuli served as the CS(-), discrimination failed due to generalization from the CS(+). Thus, the stimuli differed markedly in the specificity of conditioning. This strikingly asymmetrical stimulus generalization is a novel bias in discrimination.

Single-unit firing in rat perirhinal cortex caused by fear conditioning to arbitrary and ecological stimuli.

Pretraining lesions of rat perirhinal cortex (PR) severely impair pavlovian fear conditioning to a 22 kHz ultrasonic vocalization (USV) cue. However, PR lesions are without significant effect when the cue is a continuous tone at the same or a lower frequency. Here we examined fear-conditioning-produced changes in single-unit firing elicited in rat PR by a 22 kHz tone cue or a 22 kHz USV cue. Chronic recording electrodes were introduced from the lateral surface of the skull. Altogether, 200 well isolated units were studied in 28 rats. Overall, 73% of the recorded single units (145 of 200 units) evidenced statistically significant firing changes in response to the tone or USV conditional stimulus (CS) after it had been paired several times with an aversive unconditional stimulus (US). Interestingly, 33% of units (66 of 200 units) that were initially CS-unresponsive became CS-responsive after conditioning. After conditioning, there were two notable differences between single-unit responses elicited by the USV cue and those elicited by the tone cue. First, 11% of the units (14 of 123 units) recorded from the USV-conditioned group displayed a precisely timed increase in firing rate during the 260 ms interval in which the US had previously occurred. This US-timed response was unique to the USV-conditioned group. Second, the mean latency of cue-elicited firing was approximately 30 ms longer in the USV-conditioned group than in the tone-conditioned group. These cue-specific differences in acquired firing latencies and acquired firing patterns suggest that spectrotemporal properties of a CS can control the essential circuitry or neurophysiological mechanisms underlying fear conditioning.

Impaired trace and contextual fear conditioning in aged rats.

Trace and contextual fear conditioning were evaluated in adult (3-6 months), early middle-aged (8-12 months), late middle-aged (16-20 months), and aged (24-33 months) Sprague-Dawley rats. After trace conditioning, aged animals exhibited significantly less freezing to the tone conditioned stimulus and training context. Levels of trace-cue and context conditioning were negatively correlated with age (r = -0.56 and -0.59, respectively) and positively correlated with each other (r = +0.52). Aged rats showed robust conditioning in short- and long-delay fear paradigms, suggesting that the trace interval, rather than the use of a long interstimulus interval, is responsible for the aging-related deficits in trace fear conditioning. The authors suggest that these aging-related conditioning deficits furnish useful indices of functional changes within hippocampus or perirhinal cortex.

Neurophysiological theory of kamin blocking in fear conditioning.

Kamin blocking in fear conditioning is thought to reflect diminished processing of the unconditional stimulus (US) in the presence of a conditional stimulus (CS-super(+)) that was previously paired with this US. According to Fanselow's (1998) hypothesis, the CS-super(+) drives output from the amygdala that ultimately produces analgesia by causing opiate release onto afferent pain circuits. This hypothesis was explored quantitatively through neurophysiological simulations. The results suggest that opiate-mediated, negative-feedback control of US processing is too slow for efficient blocking of cue conditioning. The reason is that conditioning-produced synaptic modifications can be induced before the opiate-mediated inhibition has any substantial effect on US processing. The results suggest the existence of an additional, faster-acting, inhibitory neurotransmitter in the blocking circuit.

Temporal encoding in fear conditioning revealed through associative reflex facilitation.

Temporal encoding in Pavlovian fear conditioning was examined through conditional facilitation of the short-latency (Rl) component of the rat eyeblink reflex. Rats were fear-conditioned to a tone conditional stimulus (CS) with either a 3- or 9-s interstimulus interval (ISI) between CS onset and the onset of the grid-shock unconditional stimulus (US). Rl facilitation was tested over 2 days, in counterbalanced order, at a latency of 3 s and 9 s from CS onset. CS-produced Rl facilitation, the conditional response (CR), was 3-4 times larger when the test latency equaled the conditioning ISI. These results, coupled with the known neurophysiology of Rl facilitation, suggest that this CR could disclose differences in the time course of CS-generated output from the amygdala when driven by cortical versus subcortical CS-CR pathways.

Perirhinal cortex supports delay fear conditioning to rat ultrasonic social signals.

Auditory information can reach the lateral nucleus of the amygdala (LA) through a monosynaptic thalamic projection or a polysynaptic cortical route. The polymodal input from the perirhinal cortex (PR) is a major informational gateway to the LA and nearby structures. Pretraining PR lesions impair fear conditioning to a context, but there have been no reports that they cause deficits in delay conditioning to an auditory cue. The direct subcortical projection to the LA seems sufficient to support delay conditioning to a tone conditional stimulus (CS). We examined the effect of PR lesions on delay conditioning to two different tone conditional stimuli (4 and 22 kHz tones; both 10 sec duration) and two different rat ultrasonic vocalization (USV) conditional stimuli (10 sec of "22 kHz USVs"). The two USV conditional stimuli were multi-call segments that were recorded (digitized at 100 kHz) from two different rats. One USV CS was a continuous sequence of eight calls, and the other was a portion of a continuous sequence of six calls. PR lesions significantly impaired conditioning to both USV conditional stimuli and to the training context but had no significant effect on conditioning to either tone CS. The role of PR in fear conditioning appears not to be determined by whether the conditional stimuli serve as contexts or cues, but instead by the nature or complexity of the stimuli or stimulus configurations. These cue-specific effects of PR lesions are suggested to reflect differences in the stimulus features that are encoded in the two CS pathways to the LA.

Amygdalar NMDA receptors control the expression of associative reflex facilitation and three other conditional responses.

Four conditional responses (CRs) were measured in rats implanted with bilateral cannulas in the basolateral nuclear complex of the amygdala (BLA). During retention testing in either the original training context or a shifted context, BLA was infused with artificial cerebral spinal fluid (ACSF) or ACSF containing an N-methyl-D-aspartate receptor antagonist (APV). Regardless of the testing context, APV infusion into BLA completely blocked the expression of conditional eyeblink facilitation and significantly attenuated the expression of conditional freezing, ultrasonic vocalization, and defecation. Discriminant analysis found eyeblink facilitation to be comparable to freezing in predicting group membership (APV vs. ACSF) and both to be better predictors than the other two CRs. The APV effect did not depend on the exact cannula positions within BLA.

Central amygdala lesions block ultrasonic vocalization and freezing as conditional but not unconditional responses.

Bilateral amygdala (AM) lesions prevent the acquisition of fear-related conditional responses (CRs) in rats, a result that is most commonly concluded to reflect a learning or memory deficit. An alternative hypothesis is that AM-lesioned animals fail to acquire certain fear CRs simply because they cannot perform these behaviors. This performance-deficit hypothesis is usually invoked in regard to studies in which the CR is freezing, the most commonly measured behavior. Here we explore this interpretation by measuring two different behaviors [freezing and 22 kHz ultrasonic vocalization (USV)] elicited under three conditions (during context conditioning, during subsequent retention testing, and after ejaculation) in experimental rats [that received electrolytic lesions of the central nucleus of the amygdala (ACe)] and control animals (that received a sham operation). If ACe damage produces a discrete motor deficit that specifically renders the animal unable to remain immobile, then freezing should be blocked or impaired when elicited under all three conditions, whereas USV should be spared. Alternatively, if ACe damage selectively interferes with CR formation, maintenance, or expression, then both freezing and USV should be blocked or impaired when elicited as CRs during acquisition and retention testing but spared when evoked as unconditional responses (URs) to ejaculation. ACe damage blocked or severely impaired both freezing and USV elicited as CRs but had no effect on either behavior elicited as URs. We reject the motor-deficit hypothesis and discuss some viable alternatives.

Three classes of pyramidal neurons in layer V of rat perirhinal cortex.

Whole-cell recordings from 140 pyramidal neurons in layer V of rat perirhinal cortex (PR) revealed three distinct firing patterns: regular spiking (RS, 76%), burst spiking (BS, 9%), and late spiking (LS, 14%). LS neurons have not previously been reported in layer V of any cortical region. LS cells in layer V of PR exhibited delays of up to 12 s from onset of a depolarizing current step to spike threshold, followed by sustained firing. In contrast, pyramidal cells in layer V of other cortical regions contain only RS and BS cells. Within PR, the percentage of LS neurons in layer V differs markedly from what we previously observed in layers II/III (50% LS) and VI (90% LS). Morphologically, BS neurons in layer V of PR had thick primary apical dendrites that terminated in a tuft within layer I, whereas RS and LS cells had relatively thin primary apicals that terminated either diffusely or in a layer I tuft. At holding potentials near rest, PR neurons exhibited small (approximately 15 pA), inward, spontaneous postsynaptic currents (PSCs) that were indistinguishable among the three cell types. Currents evoked by minimal stimulation of layer I were about 2.8 times larger than the spontaneous PSCs. Evoked currents had unusually long onset latencies with little variation in latency, consistent with monosynaptic responses evoked by stimulation of unmyelinated fibers. The prevalence of LS cells in combination with the long-latency monosynaptically evoked PSCs suggested that PR is not a region of rapid throughput. This is consistent with anatomical data suggesting that PR is a higher-level association cortex. These data further advance an emerging picture of PR as a cortical region with a unique distribution of cell types different from other cortical regions.