Social support and fear-inhibition: an examination of underlying neural mechanisms.

Recent work has demonstrated that reminders of those we are closest to have a unique combination of effects on fear learning and represent a new category of fear inhibitors, termed prepared fear suppressors. Notably, social-support-figure images have been shown to resist becoming associated with fear, suppress conditional-fear-responding and lead to long-term fear reduction. Due to the novelty of this category, understanding the underlying neural mechanisms that support these unique abilities of social-support-reminders has yet to be investigated. Here, we examined the neural correlates that enable social-support-reminders to resist becoming associated with fear during a retardation-of-acquisition test. We found that social-support-figure-images (vs stranger-images) were less readily associated with fear, replicating prior work, and that this effect was associated with decreased amygdala activity and increased ventromedial prefrontal cortex (VMPFC) activity for social-support-figure-images (vs stranger-images), suggesting that social-support-engagement of the VMPFC and consequent inhibition of the amygdala may contribute to unique their inhibitory effects. Connectivity analyses supported this interpretation, showing greater connectivity between the VMPFC and left amygdala for social-support-figure-images (vs stranger-images).

Chronic intermittent ethanol exposure disrupts stress-related tripartite communication to impact affect-related behavioral selection in male rats.

Alcohol use disorder (AUD) is characterized by loss of intake control, increased anxiety, and susceptibility to relapse inducing stressors. Both astrocytes and neurons contribute to behavioral and hormonal consequences of chronic intermittent ethanol (CIE) exposure in animal models. Details on how CIE disrupts hypothalamic neuro-glial communication, which mediates stress responses are lacking. We conducted a behavioral battery (grooming, open field, reactivity to a single, uncued foot-shock, intermittent-access two-bottle choice ethanol drinking) followed by Ca2+ imaging in ex-vivo slices of paraventricular nucleus of the hypothalamus (PVN) from male rats exposed to CIE vapor or air-exposed controls. Ca2+ signals were evaluated in response to norepinephrine (NE) with or without selective α-adrenergic receptor (αAR) or GluN2B-containing N-methyl-D-aspartate receptor (NMDAR) antagonists, followed by dexamethasone (DEX) to mock a pharmacological stress response. Expectedly, CIE rats had altered anxiety-like, rearing, grooming, and drinking behaviors. Importantly, NE-mediated reductions in Ca2+ event frequency were blunted in both CIE neurons and astrocytes. Administration of the selective α1AR antagonist, prazosin, reversed this CIE-induced dysfunction in both cell types. Additionally, the pharmacological stress protocol reversed the altered basal Ca2+ signaling profile of CIE astrocytes. Signaling changes in astrocytes in response to NE were correlated with anxiety-like behaviors, such as the grooming:rearing ratio, suggesting tripartite synaptic function plays a role in switching between exploratory and stress-coping behavior. These data show how CIE exposure causes persistent changes to PVN neuro-glial function and provides the groundwork for how these physiological changes manifest in behavioral selection.

No effect of glucose administration in a novel contextual fear generalization protocol in rats.

The excessive transfer of fear acquired for one particular context to similar situations has been implicated in the development and maintenance of anxiety disorders, such as post-traumatic stress disorder. Recent evidence suggests that glucose ingestion improves the retention of context conditioning. It has been speculated that glucose might exert that effect by ameliorating hippocampal functioning, and may hold promise as a therapeutic add-on in traumatized patients because improved retention of contextual fear could help to restrict its generalization. However, direct data regarding the effect of glucose on contextual generalization are lacking. Here, we introduce a new behavioral protocol to study such contextual fear generalization in rats. In adult Wistar rats, our procedure yields a gradient of generalization, with progressively less freezing when going from the original training context, over a perceptually similar generalization context, to a markedly dissimilar context. Moreover, we find a flattening of the gradient when the training-test interval is prolonged with 1 week. We next examine the effect of systemic glucose administration on contextual generalization with this novel procedure. Our data do not sustain generalization-reducing effects of glucose and question its applicability in traumatic situations. In summary, we have developed a replicable contextual generalization procedure for rats and demonstrate how it is a valuable tool to examine the neurobiological correlates and test pharmacological interventions pertaining to an important mechanism in the etiology of pathological anxiety.

Inhaled anesthetic responses of recombinant receptors and knockin mice harboring α2(S270H/L277A) GABA(A) receptor subunits that are resistant to isoflurane.

The mechanism by which the inhaled anesthetic isoflurane produces amnesia and immobility is not understood. Isoflurane modulates GABA(A) receptors (GABA(A)-Rs) in a manner that makes them plausible targets. We asked whether GABA(A)-R α2 subunits contribute to a site of anesthetic action in vivo. Previous studies demonstrated that Ser270 in the second transmembrane domain is involved in the modulation of GABA(A)-Rs by volatile anesthetics and alcohol, either as a binding site or a critical allosteric residue. We engineered GABA(A)-Rs with two mutations in the α2 subunit, changing Ser270 to His and Leu277 to Ala. Recombinant receptors with these mutations demonstrated normal affinity for GABA, but substantially reduced responses to isoflurane. We then produced mutant (knockin) mice in which this mutated subunit replaced the wild-type α2 subunit. The adult mutant mice were overtly normal, although there was evidence of enhanced neonatal mortality and fear conditioning. Electrophysiological recordings from dentate granule neurons in brain slices confirmed the decreased actions of isoflurane on mutant receptors contributing to inhibitory synaptic currents. The loss of righting reflex EC(50) for isoflurane did not differ between genotypes, but time to regain the righting reflex was increased in N(2) generation knockins. This effect was not observed at the N(4) generation. Isoflurane produced immobility (as measured by tail clamp) and amnesia (as measured by fear conditioning) in both wild-type and mutant mice, and potencies (EC(50)) did not differ between the strains for these actions of isoflurane. Thus, immobility or amnesia does not require isoflurane potentiation of the α2 subunit.

Selective knockdown of NMDA receptors in primary afferent neurons decreases pain during phase 2 of the formalin test.

The role of NMDA receptors (NMDARs) expressed by primary afferent neurons in nociception remains controversial. The aim of this study was to develop mice with a tissue selective knockdown of NMDARs in these neurons and to evaluate their behavioral responses to different types of painful stimuli. Mice with floxed NMDAR NR1 subunit gene (fNR1) were crossed with mice expressing Cre recombinase under the control of the peripherin promotor (Prph-Cre). Male Prph-Cre+ floxed NR1 mice were compared to Cre- littermates. Both quantitative RT/PCR and Western blotting indicated a ∼75% reduction in NR1 expression in dorsal root ganglia (DRG) extracts with no effect on NR1 expression in spinal cord, brain or the enteric nervous system. Immunocytochemistry with antibodies to NR1 revealed decreased staining in all size classes of DRG neurons. NMDA produced a detectable increase in [Ca2+]i in 60% of DRG neurons cultured from Cre- mice, but only 15% of those from Cre+ mice. Furthermore, the peak [Ca2+]i responses were 64% lower in neurons from Cre+ mice. There was no significant difference between Cre+ and Cre- mice in response latencies to the hotplate or tail withdrawal tests of thermal nociception, nor was there a difference in withdrawal thresholds to mechanical stimuli of the tail or paw. However, compared to Cre- littermates, Cre+ knockdown mice had a 50% decrease in the phase 2 response to formalin injection (P<0.001). There was no effect on phase 1 responses. These results suggest that NMDA receptors expressed by primary afferent nerves play an important role in the development of sensitized pain states.

Trace and contextual fear conditioning is enhanced in mice lacking the alpha4 subunit of the GABA(A) receptor.

The GABA(A)R alpha4 subunit is highly expressed in the dentate gyrus region of the hippocampus at predominantly extra synaptic locations where, along with the GABA(A)R delta subunit, it forms GABA(A) receptors that mediate a tonic inhibitory current. The present study was designed to test hippocampus-dependent and hippocampus-independent learning and memory in GABA(A)R alpha4 subunit-deficient mice using trace and delay fear conditioning, respectively. Mice were of a mixed C57Bl/6J X 129S1/X1 genetic background from alpha4 heterozygous breeding pairs. The alpha4-knockout mice showed enhanced trace and contextual fear conditioning consistent with an enhancement of hippocampus-dependent learning and memory. These enhancements were sex-dependent, similar to previous studies in GABA(A)R delta knockout mice, but differences were present in both males and females. The convergent findings between alpha4 and delta knockout mice suggests that tonic inhibition mediated by alpha4betadelta GABA(A) receptors negatively modulates learning and memory processes and provides further evidence that tonic inhibition makes important functional contributions to learning and behavior.

Amygdala-dependent and amygdala-independent pathways for contextual fear conditioning.

The basolateral amygdala (BLA), consisting of the lateral and basal nuclei, is considered to be essential for fear learning. Using a temporary inactivation technique, we found that rats could acquire a context-specific long-term fear memory without the BLA but only if intensive overtraining was used. BLA-inactivated rats' learning curves were characterized by slow learning that eventually achieved the same asymptotic performance as rats with the BLA functional. BLA inactivation abolished expression of overtrained fear when rats were overtrained with a functional BLA. However, BLA-inactivation had no effect on the expression of fear in rats that learned while the BLA was inactivated. These data suggest that there are primary and alternate pathways capable of mediating fear. Normally, learning is dominated by the more efficient primary pathway, which prevents learning in the alternate pathway. However, alternate pathways compensate when the dominant pathway is compromised.

Cholinergic modulation of pavlovian fear conditioning: effects of intrahippocampal scopolamine infusion.

Cholinergic neurotransmission has been implicated in the acquisition of a variety of tasks, including Pavlovian fear conditioning. To more precisely define the role of cholinergic modulation in this process, the effect of site-specific cholinergic antagonism was assessed. Male Long-Evans rats were implanted with chronic, bilateral cannulae aimed at the dorsal hippocampus. Infusions of scopolamine hydrobromide (50 microg bilaterally) or phosphate-buffered saline (PBS) were made immediately prior to a signaled Pavlovian fear conditioning procedure. On consecutive days following training, all rats were given independent tests assessing freezing to both the training context and the tone conditional stimulus (CS). Relative to PBS infused controls, rats that received intrahippocampal infusions of scopolamine showed a significant attenuation of contextual freezing but comparable levels of freezing to the tone CS. Neither shock sensitivity nor general activity levels differed between rats infused with scopolamine or PBS. These findings suggest that fear conditioning to context, but not discrete CS, requires intact cholinergic neurotransmission in the hippocampus.

The concentration of isoflurane required to suppress learning depends on the type of learning.

BACKGROUND: Recent reports suggest that one type of learning, fear conditioning to context, requires more neural processing than a related type, fear conditioning to tone. To determine whether these types of learning were differentially affected by anesthesia, the authors applied isoflurane during the training phases of fear conditioning paradigms for freezing to context and freezing to tone. METHODS: The authors trained seven groups of eight rats to fear tone by administering a tone (conditioned stimulus) while breathing various concentrations of isoflurane from 0.00 to 0.75 minimum alveolar concentration (MAC; one concentration per group) separated by 0.12-MAC steps. On the succeeding day, and in the absence of isoflurane, the authors presented the tone (without shock) in a different context (different cage shape and odor) and measured the time each rat froze (became immobile). Six other groups of eight rats were trained to fear context by applying the shock in the absence of a tone but in the presence of environmental cues such as cage shape, texture, and odor. Fear to context was determined the succeeding day by returning the rat to the training cage (without shock) and measuring duration of freezing. Control groups (16 per group) received 0.75 MAC isoflurane but no foot shocks. Group scores were compared using analysis of variance, and the ED50 values for quantal responses of individual rats were calculated using logistic regression. RESULTS: Conditioning to context occurred at 0.00 and 0.13 MAC (P < 0.05 compared with unshocked control) but not 0.25 MAC; the ED50 was 0.25 +/- 0.03 MAC (mean +/- SEM). In contrast, conditioning to tone occurred at 0.48 MAC (P < 0.05) but not 0.62 MAC; the ED50 was 0.47 +/- 0.02 MAC (P < 0.01 for the difference between ED50 values). CONCLUSIONS: Suppression of fear conditioning to tone required approximately twice the isoflurane concentration that suppressed fear conditioning to context. Thus, the concentration of anesthetic required to suppress learning may depend on the neural substrates of learning. Our results suggest that isoflurane concentrations greater than 0.5 MAC may be needed to suppress both forms of fear conditioning.

Hippocampus and contextual fear conditioning: recent controversies and advances.

Dorsal hippocampal (DH) lesions produce a severe deficit in recently, but not remotely, acquired contextual fear without impairing memory of discrete training stimuli, i.e., DH lesions produce an anterograde and time-limited retrograde amnesia specific to contextual memory. These data are consistent with the standard model which posits temporary involvement of the hippocampus in recent memory maintenance. However, three recent controversies apparently weaken the case for a selective mnemonic role for the hippocampus in contextual fear. First, although retrograde amnesia (from posttraining lesions) is severe, anterograde amnesia (from pretraining lesions) may be mild or nonexistent. Second, a performance, rather than mnemonic, account of contextual freezing deficits in hippocampal-lesioned animals has been offered. Third, damage to the entire hippocampus, including the ventral hippocampus, can produce a dramatic and temporally stable disruption of context and tone fear. These data are reviewed and explanations are offered as to why they do not necessarily challenge the standard model of hippocampal memory function in contextual fear. Finally, a more complete description of the hippocampus' proposed role in contextual fear is offered, along with new data supporting this view. In summary, the data support a specific mnemonic role for the DH in the acquisition and consolidation of contextual representations.

Sex differences, context preexposure, and the immediate shock deficit in Pavlovian context conditioning with mice.

The acquisition of context fear in rats is affected by variables such as the sex of the animal, the placement to shock interval (PSI), and preexposure to the context. The current experiments assessed the effects of these variables on context conditioning in mice (C57BL/6). In Experiment 1, mice were placed in a chamber and received a single shock 5s, 20 s, 40s, 60s, 180s, or 720s later. Increasing the PSI produced corresponding increases in conditional freezing during the context test. In addition, male mice acquired more context conditioning than female mice did but only at intermediate PSIs. In Experiment 2, preexposure to the context before training alleviated the sex difference found with an intermediate PSI. The results are discussed in terms of configural learning theory and are argued to be contrary to the predictions of scalar expectancy theory.

Body temperature as a conditional response measure for pavlovian fear conditioning.

On six days rats were exposed to each of two contexts. They received an electric shock in one context and nothing in the other. Rats were tested later in each environment without shock. The rats froze and defecated more often in the shock-paired environment; they also exhibited a significantly larger elevation in rectal temperature in that environment. The rats discriminated between each context, and we suggest that the elevation in temperature is the consequence of associative learning. Thus, body temperature can be used as a conditional response measure in Pavlovian fear conditioning experiments that use footshock as the unconditional stimulus.

Contextual fear, gestalt memories, and the hippocampus.

This review examines the relationship between exploration and contextual fear conditioning. The fear acquired to places or contexts associated with aversive events is a form of Pavlovian conditioning. However, an initial period of exploration is necessary to allow the animal to form an integrated memory of the features of the context before conditioning can take place. The hippocampal formation plays a critical role in this process. Cells within the dorsal hippocampus are involved in the formation, storage and consolidation of this integrated representation of context. Projections from the subiculum to the nucleus accumbens regulate the exploration necessary for the acquisition of information about the features of the context. This model explains why electrolytic but not excitotoxic lesions of the dorsal hippocampus cause enhanced exploratory activity but both cause deficits in contextual fear. It also explains why retrograde amnesia of contextual fear is greater than anterograde amnesia.

Mouse model of Sanfilippo syndrome type B produced by targeted disruption of the gene encoding alpha-N-acetylglucosaminidase.

The Sanfilippo syndrome type B is an autosomal recessive disorder caused by mutation in the gene (NAGLU) encoding alpha-N-acetylglucosaminidase, a lysosomal enzyme required for the stepwise degradation of heparan sulfate. The most serious manifestations are profound mental retardation, intractable behavior problems, and death in the second decade. To generate a model for studies of pathophysiology and of potential therapy, we disrupted exon 6 of Naglu, the homologous mouse gene. Naglu-/- mice were healthy and fertile while young and could survive for 8-12 mo. They were totally deficient in alpha-N-acetylglucosaminidase and had massive accumulation of heparan sulfate in liver and kidney as well as secondary changes in activity of several other lysosomal enzymes in liver and brain and elevation of gangliosides G(M2) and G(M3) in brain. Vacuolation was seen in many cells, including macrophages, epithelial cells, and neurons, and became more prominent with age. Although most vacuoles contained finely granular material characteristic of glycosaminoglycan accumulation, large pleiomorphic inclusions were seen in some neurons and pericytes in the brain. Abnormal hypoactive behavior was manifested by 4.5-mo-old Naglu-/- mice in an open field test; the hyperactivity that is characteristic of affected children was not observed even in younger mice. In a Pavlovian fear conditioning test, the 4.5-mo-old mutant mice showed normal response to context, indicating intact hippocampal-dependent learning, but reduced response to a conditioning tone, perhaps attributable to hearing impairment. The phenotype of the alpha-N-acetylglucosaminidase-deficient mice is sufficiently similar to that of patients with the Sanfilippo syndrome type B to make these mice a good model for study of pathophysiology and for development of therapy.

Attenuated sensitivity to neuroactive steroids in gamma-aminobutyrate type A receptor delta subunit knockout mice.

gamma-Aminobutyric acid (GABA) type A receptors mediate fast inhibitory synaptic transmission and have been implicated in responses to sedative/hypnotic agents (including neuroactive steroids), anxiety, and learning and memory. Using gene targeting technology, we generated a strain of mice deficient in the delta subunit of the GABA type A receptors. In vivo testing of various behavioral responses revealed a strikingly selective attenuation of responses to neuroactive steroids, but not to other modulatory drugs. Electrophysiological recordings from hippocampal slices revealed a significantly faster miniature inhibitory postsynaptic current decay time in null mice, with no change in miniature inhibitory postsynaptic current amplitude or frequency. Learning and memory assessed with fear conditioning were normal. These results begin to illuminate the novel contributions of the delta subunit to GABA pharmacology and sedative/hypnotic responses and behavior and provide insights into the physiology of neurosteroids.

Anxiety: at the intersection of genes and experience.

Human anxiety disorders arise from a combination of genetic vulnerability and traumatic experience. Mice with a GABAA receptor mutation may provide a model for these disorders.

The neuroanatomical and neurochemical basis of conditioned fear.

After a few pairings of a threatening stimulus with a formerly neutral cue, animals and humans will experience a state of conditioned fear when only the cue is present. Conditioned fear provides a critical survival-related function in the face of threat by activating a range of protective behaviors. The present review summarizes and compares the results of different laboratories investigating the neuroanatomical and neurochemical basis of conditioned fear, focusing primarily on the behavioral models of freezing and fear-potentiated startle in rats. On the basis of these studies, we describe the pathways mediating and modulating fear. We identify several key unanswered questions and discuss possible implications for the understanding of human anxiety disorders.

Temporally graded retrograde amnesia of contextual fear after hippocampal damage in rats: within-subjects examination.

We have shown previously that electrolytic lesions of the dorsal hippocampus (DH) produce a severe deficit in contextual fear if made 1 d, but not 28 d, after fear conditioning (). As such, the hippocampus seems to play a time-limited role in the consolidation of contextual fear conditioning. Here, we examine retrograde amnesia of contextual fear produced by DH lesions in a within-subjects design. Unlike our previous reports, rats had both a remote and recent memory at the time of the lesion. Rats were given 10 tone-shock pairings in one context (remote memory) and 10 tone-shock pairings in a distinct context (with a different tone) 50 d later (recent memory), followed by DH or sham lesions 1 d later. Relative to controls, DH-lesioned rats exhibited no deficit in remote contextual fear, but recent contextual fear memory was severely impaired. They also did not exhibit deficits in tone freezing. This highly specific deficit in recent contextual memory demonstrated in a within-subjects design favors mnemonic over performance accounts of hippocampal involvement in fear. These findings also provide further support for a time-limited role of the hippocampus in memory storage.

Scopolamine and Pavlovian fear conditioning in rats: dose-effect analysis.

Muscarinic-cholinergic antagonism produces learning and memory deficits in a wide variety of hippocampal-dependent tasks. Hippocampal lesions produce both acquisition deficits and retrograde amnesia of contextual fear (fear of the place of conditioning), but do not impact fear conditioning to discrete cues (such as a tone). In order to examine the effects of muscarinic antagonism in this paradigm, rats were given 0.01 to 100 mg/kg of scopolamine (or methylscopolamine) either before or after a fear conditioning session in which tones were paired with aversive footshocks. Fear to the context and the tone were assessed by measuring freezing in separate tests. It was found that pretraining, but not post-training, scopolamine severely impaired fear conditioning; methylscopolamine was ineffective in disrupting conditioning. Although contextual fear conditioning was more sensitive to cholinergic disruption, high doses of scopolamine also disrupted tone conditioning. Scopolamine did not affect footshock reactivity, but did produce high levels of activity. However, hyperactivity was not directly responsible for deficits in conditioning. It was concluded that scopolamine disrupts CS-US association formation or CS processing, perhaps through an attenuation of hippocampal theta rhythm.