Diazepam promotes active avoidance extinction associating with increased dorsal CA3 and amygdala activity.

Active avoidance (AA) is an adaptive response to potentially harmful situations while maladapted avoidance that does not extinguish is one of the core symptoms of anxiety and post-traumatic stress disorder. However, the neural mechanisms of AA extinction and its relationship to anxiety remain unclear. We examined AA extinction during three extinction training sessions in two-way active avoidance paradigm and tested the effect of anxiolytic on AA extinction. Then we performed a meta-analysis of rodent studies, identified anxiolytic diazepam facilitates AA acquisition, and tested the same treatment in AA extinction. Diazepam-treated rats significantly reduced avoidance in the first two extinction training, compared with the saline-treated rats, and the reduction in avoidance remained in the third drug-free session. Then we explored extinction associated hippocampal and amygdala activity in saline-and diazepam-treated rats using c-Fos immunostaining following the last extinction session. The density of c-Fos positive cells was higher in dorsal CA3 of the diazepam group than in that of saline-treated animals, and was also higher in the central and basolateral amygdala regions of diazepam-treated rats than in that of saline-treated animals. Combined, these results suggest anxiolytics promotes AA extinction associated with dorsal CA3 and amygdala activity changes.

Hippocampal injury and learning deficits following non-convulsive status epilepticus in periadolescent rats.

The effects of non-convulsive status epilepticus (NCSE) on the developing brain remain largely elusive. Here we investigated potential hippocampal injury and learning deficits following one or two episodes of NCSE in periadolescent rats. Non-convulsive status epilepticus was induced with subconvulsive doses of intrahippocampal kainic acid (KA) under continuous EEG monitoring in postnatal day 43 (P43) rats. The RKA group (repeated KA) received intrahippocampal KA at P43 and P44, the SKA group (single KA injection) received KA at P43 and an intrahippocampal saline injection at P44. Controls were sham-treated with saline. The modified two-way active avoidance (MAAV) test was conducted between P45 and P52 to assess learning of context-cued and tone-signaled electrical foot-shock avoidance. Histological analyses were performed at P52 to assess hippocampal neuronal densities, as well as potential reactive astrocytosis and synaptic dysfunction with GFAP (glial fibrillary acidic protein) and synaptophysin (Syp) staining, respectively. Kainic acid injections resulted in electroclinical seizures characterized by behavioral arrest, oromotor automatisms and salivation, without tonic-clonic activity. Compared to controls, both the SKA and RKA groups had lower rates of tone-signaled shock avoidance (p < 0.05). In contextual testing, SKA rats were comparable to controls (p > 0.05), but the RKA group had learning deficits (p < 0.05). Hippocampal neuronal densities were comparable in all groups. Compared to controls, both the SKA and RKA groups had higher hippocampal GFAP levels (p < 0.05). The RKA group also had lower hippocampal Syp levels compared to the SKA and control groups (p < 0.05), which were comparable (p > 0.05). We show that hippocampal NCSE in periadolescent rats results in a seizure burden-dependent hippocampal injury accompanied by cognitive deficits. Our data suggest that the diagnosis and treatment of NCSE should be prompt.

A Modified Two-Way Active Avoidance Test for Combined Contextual and Auditory Instrumental Conditioning.

Available two-way active avoidance paradigms do not provide contextual testing, likely due to challenges in performing repetitive trials of context exposure. To incorporate contextual conditioning in the two-way shuttle box, we contextually modified one of the chambers of a standard two-chamber rat shuttle box with visual cues consisting of objects and black and white stripe patterns. During the 5 training days, electrical foot shocks were delivered every 10 s in the contextually modified chamber but were signaled by a tone in the plain chamber. Shuttling between chambers prevented an incoming foot shock (avoidance) or aborted an ongoing one (escape). During contextual retention testing, rats were allowed to freely roam in the box. During auditory retention testing, visual cues were removed, and tone-signaled shocks were delivered in both chambers. Avoidance gradually replaced escape or freezing behaviors reaching 80% on the last training day in both chambers. Rats spent twice more time in the plain chamber during contextual retention testing and had 90% avoidance rates during auditory retention testing. Our modified test successfully assesses both auditory and contextual two-way active avoidance. By efficiently expanding its array of outcomes, our novel test will complement standard two-way active avoidance in mechanistic studies and will improve its applications in translational research.

Effects of a methanol extract of Ficus platyphylla stem bark on a two-way active avoidance task and on body core temperature.

Preparations of Ficus platyphylla are used in Nigeria's folk medicine to manage a variety of diseases, including insomnia, psychoses, depression, epilepsy, pain, and inflammation. In this study, we examined the effects of the standardised methanol extract of F. platyphylla stem bark (FP) on two-way active avoidance learning and body core temperature to complement earlier studies on the neuroleptic potential of this medicinal plant, which is already in common use. The extract did not interfere with the acquisition and consolidation of the conditioned avoidance reaction (CAR), but did diminish the retrieval of CAR. The extract dose-dependently reduced body core temperature; this was significantly ameliorated by the use of amphetamine. The results confirmed the neuroleptic-like efficacy of FP, probably via the modulation of dopaminergic neurons.

High fat diet increases cognitive decline and neuroinflammation in a model of orexin loss.

Midlife obesity is a risk factor for cognitive decline and is associated with the earlier onset of Alzheimer's disease (AD). Diets high in saturated fat potentiate the onset of obesity, microglial activation, and neuroinflammation. Signaling deficiencies in the hypothalamic peptide orexin and/or orexin fiber loss are linked to neurodegeneration, cognitive impairment, and neuroinflammation. Prior studies show that orexin is neuroprotective, suppresses neuroinflammation, and that treatment with orexin improves cognitive processes in orexin/ataxin-3 (O/A3) mice, a transgenic mouse model of orexin neurodegeneration. Our overall hypothesis is that loss of orexin contributes to high fat diet (HFD)-induced hippocampal neuroinflammation and cognitive decline. To examine this, we tested male O/A3 mice (7-8 mo. of age) in a two-way active avoidance (TWAA) hippocampus-dependent memory task. We tested whether (1) orexin loss impaired cognitive function; (2) HFD worsened cognitive impairment; and (3) HFD increased microglial activation and neuroinflammation. O/A3 mice showed significant impairments in TWAA task learning vs. wild type (WT) mice (increased escapes p < 0.05, reduced avoidances p < 0.0001). Mice were then placed on HFD (45% total fat, 31.4% saturated fat) or remained on normal chow (NC; 4% total fat and 1% saturated fat), and TWAA was retested at 2 and 4 weeks. Learning impairment was evident at both 2 and 4 weeks in O/A3 mice fed HFD for following diet exposure vs. WT mice on normal chow or HFD (increased escapes, reduced avoidances p < 0.05). Additionally, O/A3 mice had increased gene expression of the microglial activation marker Iba-1 (measured via qRT-PCR, p < 0.001). Further characterization of the microglial immune response genes in hippocampal tissue revealed a significant increase in CX3 chemokine receptor 1 (CX3CR1), tumor necrosis factor-alpha (TNF-α) and the mitochondria-associated enzyme immune responsive gene-1 (Irg1). Collectively, our results indicate that orexin loss impairs memory, and that HFD accelerates hippocampus-dependent learning deficits and the onset of neuroinflammation.

Active avoidance learning differentially activates ERK phosphorylation in the primary auditory and visual cortices of Roman high- and low-avoidance rats.

Understanding the mechanisms underlying conditioned avoidance is a critical step toward the development of novel treatments of anxiety. In this context, the two-way active avoidance (2WAA) task is a validated paradigm to investigate uncontrolled avoidance, a hallmark of anxiety disorders. The outbred Roman high- (RHA) and low-avoidance (RLA) rat lines are selected for respectively rapid vs. poor acquisition of active avoidant behavior, and emotional reactivity appears to be the most prominent behavioral difference between the two lines, with RLA rats being more fearful/anxious than their RHA counterparts. This study was aimed at assessing the relationship between the different performance of RHA and RLA rats in the 2WAA task and the number of phosphorylated ERK positive (pERK+) neurons in the primary auditory and visual cortices, in three sub-nuclei of the amygdala, as well as in the nucleus accumbens, and the prefrontal cortex. The results indicate that: (1) RHA rats, but not their RLA counterparts, learn very rapidly to avoid mild electric foot-shocks by crossing to the opposite compartment of the shuttle-box during the presentation of the conditioned stimulus and (2) the different behavior of the Roman lines during active avoidance training is associated with differential changes in the number of pERK+ neurons in the primary auditory and visual cortices (where the proactive coping of RHA rats is associated with increased ERK phosphorylation), but not in the other brain areas examined. These results are consistent with the hypothesis that the activation of the ERK signaling cascade in the auditory and visual cortices may be involved in the acquisition of aversive learning in RHA rats.

Spaced sessions of avoidance extinction reduce spontaneous recovery and promote infralimbic cortex activation.

Extinction-based therapies (EBT) are the psychological treatments of choice for certain anxiety disorders, such as post-traumatic stress disorder. However, some patients relapse and suffer spontaneous recovery (SR) of anxiety symptoms and persistence of avoidance behaviour, which underlines the need for improving EBT. In rats, recent evidence has highlighted the relevance of the temporal distribution of extinction sessions in reducing SR of auditory fear conditioning, although it has seldom been studied in procedures involving proactive avoidance responses, such as two-way active avoidance conditioning (TWAA). We examined whether the temporal distribution of two extinction sessions separated by 24h or 7days (contiguous versus spaced extinction paradigms, respectively), influences SR after 28days of a TWAA task. c-Fos expression, as a marker of neuronal activation, was also measured by immunohistochemistry 90min after the SR test in the amygdala and the medial prefrontal cortex. The temporal distribution of extinction sessions did not affect the degree of extinction learning. However, only the rats that underwent the 7-day spaced extinction paradigm maintained the level of extinction in the long term, showing no SR in TWAA. This behavioural finding was consistent with a greater number of c-Fos-labelled neurons in the infralimbic cortex in the 7-day group, and in the Lateral and Central nuclei of the amygdala in the 24-hour group. These findings show that a time-spaced extinction paradigm reduces the spontaneous recovery of active avoidance behaviour, and that this behavioural advantage appears to be related to the activation of the infralimbic cortex.

Gene expression analysis of Arc mRNA as a neuronal cell activity marker in the hippocampus and amygdala in two-way active avoidance test in rats.

INTRODUCTION: Immediate early genes are widely used as neuronal cell activity markers in neuroscience. The present study investigated the relationship between their expression and abnormality in context fear conditioning. METHODS: The learning test (two-way active avoidance test) was conducted in male rats administered with nonselective muscarinic antagonist scopolamine or selective dopamine D1-like receptor antagonist SCH 23390 at a dose level of 2.0 or 0.1mg/kg, respectively, for 4days. Expression levels of Arc and Fos mRNA in the hippocampus and amygdala were also evaluated on the second day of dosing by fluorescent in situ hybridization (FISH) and reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR). RESULTS: Scopolamine had no effect on avoidance rate, but decreased freezing in the two-way active avoidance test. SCH 23390 decreased avoidance rate and increased freezing. In FISH and RT-qPCR assays, scopolamine decreased Arc mRNA in the hippocampus and amygdala, whereas SCH 23390 increased Arc mRNA in the hippocampus. By contrast, scopolamine and SCH 23390 did not change Fos mRNA expression compared to Arc mRNA expression. DISCUSSION: The results of the learning test indicated that scopolamine or SCH 23390 respectively inhibited fear or context conditioning in rats. Furthermore, alteration of the expression of Arc mRNA but not of Fos mRNA in the hippocampus and amygdala of the brain was suggested to be a sensitive neuronal cell activity marker to detect behavioral abnormality in the two-way active avoidance test.

Conservation of Phenotypes in the Roman High- and Low-Avoidance Rat Strains After Embryo Transfer.

The Roman high- (RHA-I) and low-avoidance (RLA-I) rat strains are bi-directionally bred for their good versus non-acquisition of two-way active avoidance, respectively. They have recently been re-derived through embryo transfer (ET) to Sprague-Dawley females to generate specific pathogen free (SPF) RHA-I/RLA-I rats. Offspring were phenotyped at generations 1 (G1, born from Sprague-Dawley females), 3 and 5 (G3 and G5, born from RHA-I and RLA-I from G2-G4, respectively), and compared with generation 60 from our non-SPF colony. Phenotyping included two-way avoidance acquisition, context-conditioned fear, open-field behaviour, novelty-seeking, baseline startle, pre-pulse inhibition (PPI) and stress-induced increase in plasma corticosterone concentration. Post-ET between-strain differences in avoidance acquisition, context-conditioned freezing and novelty-induced self-grooming are conserved. Other behavioural traits (i.e. hole-board head-dipping, novel object exploration, open-field activity, startle, PPI) differentiate the strains at G3-G5 but not at G1, suggesting that the pre-/post-natal environment may have influenced these co-segregated traits at G1, though further selection pressure along the subsequent generations (G1-G5) rescues the typical strain-related differences.

Lesions of the lateral habenula facilitate active avoidance learning and threat extinction.

The lateral habenula (LHb) is an epithalamic brain structure that provides strong projections to midbrain monoaminergic systems that are involved in motivation, emotion, and reinforcement learning. LHb neurons are known to convey information about aversive outcomes and negative prediction errors, suggesting a role in learning from aversive events. To test this idea, we examined the effects of electrolytic lesions of the LHb on signaled two-way active avoidance learning in which rats were trained to avoid an unconditioned stimulus (US) by taking a proactive shuttling response to an auditory conditioned stimulus (CS). The lesioned animals learned the avoidance response significantly faster than the control groups. In a separate experiment, we also investigated whether the LHb contributes to Pavlovian threat (fear) conditioning and extinction. Following paired presentations of the CS and the US, LHb-lesioned animals showed normal acquisition of conditioned response (CR) measured with freezing. However, extinction of the CR in the subsequent CS-only session was significantly faster. The enhanced performance in avoidance learning and in threat extinction jointly suggests that the LHb normally plays an inhibitory role in learning driven by absence of aversive outcomes.