Extinction deficit and fear reinstatement after electrical stimulation of the amygdala: implications for kindling-associated fear and anxiety

Generalized seizures produced by electrical kindling of the amygdala in laboratory rats are a widely used animal model of temporal lobe epilepsy. In addition to seizure evolution amygdala kindling enhances emotionality. The relative roles of electrical stimulation and seizure induction in fear responding are unclear. Here we investigate this issue using extinction and reinstatement of fear-potentiated startle. After classical conditioning (light+footshock pairings) laboratory rats were fear extinguished with each light presentation followed by nonepileptogenic amygdala stimulation. In contrast to the normal extinction learning of control subjects, amygdala stimulated animals exhibited conditioned fear after 120 presentations of the nonreinforced conditioned stimulus (CS). In a second experiment electrical stimulation of the amygdala restored extinguished fear responding and the fear reinstatement was specific to extinction context. The reinstatement effect did not involve sensitized fear to the CS produced by amygdala stimulation. The possibility that electrical activation of the amygdala produces unconditioned fear was considered. Animals uniformly failed to demonstrate fear-potentiated startle using electrical stimulation of the amygdala as the unconditioned stimulus. This was the case with a subthreshold afterdischarge stimulus and a stimulation schedule that produced kindled seizures. The extinction deficit and fear reinstatement results were interpreted to suggest that amygdala stimulation activates acquired excitatory stimulus-affect neural connections formed during Pavlovian fear conditioning. Our data supports a model in which excitation of an amygdala-based memory-retrieval system reinforces the expression of learned fear behaviors.     

Involvement of norepinephrine in startle arousal after acute and chronic d-amphetamine administration

Treatment with d-amphetamine produced a dose-dependent increase in startle amplitude in response to a buzzer. This increase appeared to be a reflection of a sensitization effect, i.e., enhanced responsivity as a function of repeated stimulus presentations. Treatment with -methyl-p-tyrosine, which reduced whole brain concentrations of dopamine (DA) and norepinephrine (NE), or treatment with FLA-63, which reduced only NE, antagonized the effects of d-amphetamine on the startle reflex, suggesting a role of NE in this behavior. Startle amplitude was also reduced following chronic d-amphetamine treatment. The effect of d-amphetamine on startle was found to be independent of changes in drug-induced locomotor excitation. The data of the present investigation, together with earlier reports, suggests that tolerance occurs to those behaviors that involve a noradrenergic component.     

Response sensitization and depression following long-term amphetamine treatment in a self-stimulation paradigm

The effects of long-term amphetamine treatment were evaluated on responding supported by self-stimulation of the substantia nigra. Rats repeatedly treated with d-amphetamine, and tested with a low dose of the drug that ordinarily has no behavioral effect, showed higher response rates than animals repeatedly treated with saline and tested with the same dose of amphetamine. In contrast, a depression in responding was observed among animals that received long-term amphetamine administration and were tested with saline. The effects of long-term amphetamine treatment on self-stimulation could not be explained by the intrusion of drug-induced competitive behaviors such as locomotor activity and stereotypy. The results were attributed to changes in dopamine neurotransmission following prolonged exposure to amphetamine and were also discussed in terms of an animal model for amphetamine psychosis and postamphetamine depression in man.     

Circling behavior following systemic d-amphetamine administration: Potential noradrenergic and dopaminergic involvement

Systemic treatment with d-amphetamine produced a dose-dependent increase in the circling behavior of normal mice. Treatment with both -methyl-p-tyrosine (-MpT) and FLA-63 antagonized the amphetamine-induced circling behavior. Similarly, blockade of B-adrenergic receptors by propranolol and dopamine receptors by haloperidol reversed the circling response elicited by amphetamine. In contrast to -MpT and haloperidol, however, neither FLA-63 nor propranolol attenuated the locomotor excitation engendered by amphetamine. Following repeated d-amphetamine injections the circling ordinarily induced by a single injection was abolished, whereas the locomotor effects of amphetamine remained unaltered. These findings are consistent with earlier work suggesting that tolerance may occur in those behaviors that involve a noradrenergic component.     

Sex-specific impact of severe obesity in the outcomes of hospitalized patients with COVID-19: a large retrospective study from the Bronx,New York

It has been demonstrated that obesity is an independent risk factor for worse outcomes in patients with COVID-19. Our objectives were to investigate which classes of obesity are associated with higher in-hospital mortality and to assess the association between obesity and systemic inflammation. This was a retrospective study which included consecutive hospitalized patients with COVID-19 in a tertiary center. Three thousand five hundred thirty patients were included in this analysis (female sex: 1579, median age: 65 years). The median body mass index (BMI) was 28.8 kg/m². In the overall cohort, a J-shaped association between BMI and in-hospital mortality was depicted. In the subgroup of men, BMI 35–39.9 kg/m² and BMI ≥40 kg/m² were found to have significant association with higher in-hospital mortality, while only BMI ≥40 kg/m² was found significant in the subgroup of women. No significant association between BMI and IL-6 was noted. Obesity classes II and III in men and obesity class III in women were independently associated with higher in-hospital mortality in patients with COVID-19. The male population with severe obesity was the one that mainly drove this association. No significant association between BMI and IL-6 was noted.

     

Dabigatran,rivaroxaban, and apixaban are superior to warfarin in Asian patients with non-valvular atrial fibrillation: An updated meta-analysis

BACKGROUNDMost of the randomized clinical trials that led to the wide use of non-vitamin K antagonist oral anticoagulants for stroke prevention in patients with atrial fibrillation (AF) originated from western countries. AIMTo systematically review and quantitatively synthesize the real-world data regarding the efficacy and safety of dabigatran, rivaroxaban, and apixaban compared to warfarin for stroke prevention in Asian patients with non-valvular AF.METHODSMedline, Cochrane, and ClinicalTrial.gov databases were reviewed. A random-effect model meta-analysis was used and I-square was utilized to assess the heterogeneity. The primary outcome was ischemic stroke. The secondary outcomes were all-cause mortality, major bleeding, intracranial hemorrhage, and gastrointestinal bleeding.RESULTSTwelve studies from East Asia or Southeast Asia and 441450 patients were included. Dabigatran, rivaroxaban, and apixaban were associated with a significant reduction in the incidence of ischemic stroke [hazard ratio (HR) = 0.78, 95% confidence interval (CI): 0.65-0.94; HR = 0.79, 95%CI: 0.74-0.85, HR = 0.70, 95%CI: 0.62-0.78; respectively], all-cause mortality (HR = 0.68, 95%CI: 0.56-0.83; HR = 0.66, 95%CI: 0.52-0.84; HR = 0.66, 95%CI: 0.49-0.90; respectively), and major bleeding (HR = 0.61, 95%CI: 0.54-0.69; HR = 0.70, 95%CI: 0.54-0.90; HR = 0.58, 95%CI: 0.43-0.78; respectively) compared to warfarin.CONCLUSIONDabigatran, rivaroxaban, and apixaban appear to be superior to warfarin in both efficacy and safety in Asians with non-valvular AF.     

Structural plasticity of 4-α-helical bundles exemplified by the puzzle-like molecular assembly of the Rop protein

The dimeric Repressor of Primer (Rop) protein, a widely used model system for the study of coiled-coil 4-α-helical bundles, is characterized by a remarkable structural plasticity. Loop region mutations lead to a wide range of topologies, folding states, and altered physicochemical properties. A protein-folding study of Rop and several loop variants has identified specific residues and sequences that are linked to the observed structural plasticity. Apart from the native state, native-like and molten-globule states have been identified; these states are sensitive to reducing agents due to the formation of nonnative disulfide bridges. Pro residues in the loop are critical for the establishment of new topologies and molten globule states; their effects, however, can be in part compensated by Gly residues. The extreme plasticity in the assembly of 4-α-helical bundles reflects the capacity of the Rop sequence to combine a specific set of hydrophobic residues into strikingly different hydrophobic cores. These cores include highly hydrated ones that are consistent with the formation of interchain, nonnative disulfide bridges and the establishment of molten globules. Potential applications of this structural plasticity are among others in the engineering of bio-inspired materials.Recurrent motifs of tertiary structure are convenient model systems for studying protein folding and potentially also for the design of bio-inspired materials. For protein design purposes, structural plasticity is an important, although poorly understood, parameter to be considered, as it is among the main reasons that the re-engineering of proteins toward novel materials is not yet satisfactorily manageable (1, 2).The present study focuses on the structural plasticity associated with the 4-α-helical bundle (4HB) motif. 4HBs consist of four amphipathic α-helices packed in a parallel or antiparallel fashion (3, 4). Their folding is largely determined by a repeating pattern of hydrophobic and hydrophilic residues, organized on the basis of seven-residue repeats (heptads) (5). Being the simplest tertiary motif, 4HBs have been subject to numerous protein-folding studies; attempts have been made to exploit them as building blocks for bio-inspired materials (6).A paradigm of a highly regular 4HB is the RNA-binding ColE1 Repressor of Primer (Rop) protein (7–9), also referred to as RNA-one-modulator (ROM). Each monomer is an α-helical hairpin consisting of two antiparallel α-helices connected by a short loop. The sequence of Rop displays a heptad repeats pattern that is interrupted only in the loop region.Structural simplicity makes Rop an attractive model system for the study of the folding of 4HBs. The loop region and the hydrophobic core have thereby attracted particular attention, as these regions are linked with the remarkable ability of Rop mutants to adopt altered topologies and properties (10–15). Striking examples of loop variants include mutant Loopless Rop (LLR), in which an uninterrupted pattern of heptad repeats is established through a five-residue deletion in the loop. In this “loopless” mutant, the α-helical hairpin of the monomer is converted into a single helix (15, 16). The complete LLR molecule is a tetramer that is completely reorganized relative to the dimeric wild-type (WT) Rop, thereby becoming a hyper-thermostable protein (16). On the other hand, establishment of an uninterrupted heptad periodicity through a two-residue insertion in the loop produces minimal changes relative to WT in terms of structure and properties (12). Thus, these two mutants with uninterrupted patterns of heptads reveal that there is a considerable structural plasticity inherent to the Rop sequence, but the relationship between heptad periodicity and the structural/physicochemical properties is complex.Extreme structural plasticity producing completely altered 4HB topologies is also associated with point mutations in the loop region. Replacement of loop residue Ala31 by Pro (17) results in a complete reorganization of the entire protein, which is converted from the canonical left-handed, all-antiparallel form into a right-handed mixed-parallel and antiparallel 4-α-helical bundle, displaying a “bisecting U” topology that is to a large extent determined by the local conformation at residue 31 (18). Mutant A31P displays two variations of the bisecting U topology; these differ in the relative juxtaposition of the α-helices (19). These conformations crystallize in different space groups (orthorhombic and monoclinic); both space groups have been occasionally observed in the same crystallization drop, indicating the coexistence of the two forms in solution. Molecular dynamics simulations for A31P have demonstrated a potential for the interconversion between the two conformations (14).Hydrophobic core mutants occasionally also display structural plasticity producing a new (“syn”) topology (20) that results from the “anti”-topology of WT through a 180° flip of one monomer around the dyad axis normal to the long axis of WT. The competition between the anti and syn topologies and the mixture of the two structures have been studied in detail for some Rop mutants (21, 22).Apart from structural plasticity, a closely related issue associated with the folding of Rop is the role of Cys residues (Cys-38 and Cys-52). Both residues are buried in the hydrophobic core and are not involved in the formation of disulfide bridges in any of the known structures of WT and its mutants. Surprisingly, however, a Cys-free variant (CYSfree) that conserves the structure, stability, and in vivo activity of WT exhibits dramatically faster unfolding kinetics (23).The present study focuses on the role of the loop region and Cys residues in the structural plasticity of Rop. To explore the conversion of the WT anti-topology into the bisecting U topology of A31P, the three double mutants D30P/A31G (PG), D30G/A31P (GP), and D30P/A31P (PP) have been constructed for loop positions 30 and 31. These mutations combine the effects of the most constrained amino acid (Pro) and of the least constrained one (Gly). In addition, the potential role of Cys residues in Rop folding is explored by following the effects of reducing agents.     

Amphetamine-elicited perseverative and rotational behavior: evaluation of directional preference

Four experiments examined the effects of d-amphetamine on lateralized direction preference, circling behavior, and perseveration. The nature as well as the magnitude of the behavioral response to amphetamine was dependent upon the testing situation. Whereas 5.0-10.0 mg/kg of the drug induced a robust circling behavior in a circular alleyway, it required 10.0 mg/kg of amphetamine to produce a significant turn preference in an open field exploratory situation. In a symmetrical Y-maze, mice displayed spontaneous alternation behavior characterized by an exploratory pattern involving different arm sequences. After amphetamine administration, spontaneous alternation was reduced and pronounced perseverative patterns of exploration were evident at dose levels similar to those that induced rotational behavior. Directional preferences could not account for the exploratory patterns in the Y-maze of animals in the undrugged state, or under the influence of amphetamine regardless of dose. Since lateralized motor asymmetries after amphetamine were limited by the behavioral context in which the effects of the drug were evaluated, it was argued that rotational behavior cannot be considered to be a simple mechanistic behavior. Rather, in addition to the expression of a side preference, it involves a drug-induced perseverative tendency. On the basis of these data, it was suggested that evaluation of the direction of turning in an open field is a less biased measure of spatial preference following amphetamine administration. It was further argued that perseverative behavior is a prepotent response to amphetamine and the possibility of lateralized attentional asymmetries was discussed.     

Behavioral evidence implicating dopamine in sensorimotor arousal and norepinephrine in the sedative effects of antidepressant drugs

The effects of acute and chronic antidepressant treatment on acoustic startle were evaluated in three experiments. Administration of 2.5–10.0 mg/kg desipramine, amitriptyline, and nortriptyline depressed acoustic startle responding after repeated sensory stimulation. In contrast to the tricyclic drugs, the serotonin reuptake inhibitor zimelidine increased acoustic startle, and inhibition of dopamine reuptake following acute nomifensine and bupropion administration did not influence startle reactivity in the doses examined. The response reducing effects of desipramine and amitriptyline persisted following chronic exposure to these drugs, and these findings were discussed in relation to the inhibitory actions of the tricyclics on locus coeruleus neurons. A second major finding in this study was that animals challenged withd-amphetamine during desipramine and amitriptyline withdrawal showed a facilitated startle response. Enhanced startle reactivity to amphetamine was also observed following long-term exposure to iprindole, and a withdrawal hyperactivity of acoustic startle was evident after chronic treatment with amoxapine, bupropion, and nomifensine. These results agree with evidence that repeated administration of antidepressants increases dopamine neurotransmission which modulates sensorimotor arousal.     

Attenuation of perseverative behavior after repeated amphetamine treatment: Tolerance or attentional deficits?

When permitted to explore an 8-arm radial maze, animals exhibited a systematic pattern of exploration characterized by preference for the most novel arms (spontaneous alternation) and entry into immediately adjacent arms (adjacent alternation). Acute treatment with moderate dosages of amphetamine reduced the proportion of both types of alternation responses and induced marked stimulus perseveration, i.e., consecutive entries between pairs of arms. Prior exposure to the apparatus enhanced the degree of perseveration ordinarily observed, and provoked perseveration after low doses of the drug. In contrast to acute drug treatment, perseveration was reduced after chronic amphetamine administration. However, chronic amphetamine treatment did not appear simply to reduce the potency of the drug. In contrast to the effects of apparatus pre-exposure on the degree of perseveration induced by acute amphetamine treatment, the degree of perseveration was not enhanced by pre-exposure to the maze in mice with a history of chronic amphetamine administration. Moreover, the exploratory pattern evident in chronically treated animals differed from that of control animals even when tested in the nondrug state. That is, animals chronically treated with amphetamine and tested with saline exhibited alternation scores which did not deviate from chance. These data suggest that chronic amphetamine treatment alters the way in which organisms attend, or respond, to environmental stimuli.