Behavioral manifestations in rodent models of autism spectrum disorder: protocol for a systematic review and network meta-analysis.

BACKGROUND: Autism spectrum disorder (ASD) is a neurodevelopmental condition associated with severe social communication, interaction, and sensory processing impairments. Efforts to understand its etiology and pathophysiology are crucial for improving treatment and prevention measures. Preclinical models of ASD are essential for investigating the biological mechanisms and should present translatability potential. We aim to evaluate the consistency of the most commonly used rodent models of ASD in displaying autistic-like behavior through a systematic review and meta-analysis. METHODS: This review will focus on the most frequently used autism models, surveying studies of six genetic (Ube3a, Pten, Nlgn3, Shank3, Mecp2, and Fmr1), three chemically induced (valproic acid (VPA), lipopolysaccharide (LPS), and polyinosinic:polycytidylic acid (poly(I:C))), and one inbred model (BTBR T+ Itpr3tf/J mouse strain). Two independent reviewers will screen the records. Data extraction of behavioral outcomes and risk of bias evaluation will be performed. We will conduct a meta-analysis whenever at least five studies investigate the same model and behavioral outcome. We will also explore the heterogeneity and publication bias. Network meta-analyses are planned to compare different models. DISCUSSION: By shortening the gap between animal behavior and human endophenotypes or specific clinical symptoms, we expect to help researchers on which rodent models are adequate for research of specific behavioral manifestations of autism, which potentially require a combination of them depending on the research interest. SYSTEMATIC REVIEW REGISTRATION: PROSPERO CRD42021226299 .

Female but not male rats show biphasic effects of low doses of Δ9-tetrahydrocannabinol on anxiety: can cannabidiol interfere with these effects?

Δ9-tetrahydrocannabinol (THC) is the main phytocannabinoid present in the Cannabis sativa. It can produce dose-dependent anxiolytic or anxiogenic effects in males. THC effects on anxiety have scarcely been studied in females, despite their higher prevalence of anxiety disorders. Cannabidiol, another phytocannabinoid, has been reported to attenuate anxiety and some THC-induced effects. The present study aimed to investigate the behavioral and neurochemical effects of THC administered alone or combined with CBD in naturally cycling female rats tested in the elevated plus-maze. Systemically administered THC produced biphasic effects in females, anxiolytic at low doses (0.075 or 0.1 mg/kg) and anxiogenic at a higher dose (1.0 mg/kg). No anxiety changes were observed in males treated with the same THC dose range. The anxiogenic effect of THC was prevented by co-administration of CBD (1.0 or 3.0 mg/kg). CBD (3.0 mg/kg) caused an anxiolytic effect. At a lower dose (1.0 mg/kg), it facilitated the anxiolytic effect of the low THC dose. The anxiogenic effect of THC was accompanied by increased dopamine levels in the medial prefrontal cortex (mPFC) and nucleus accumbens (NAc). In contrast, its anxiolytic effect was associated with increased mPFC serotonin concentrations. The anxiolytic effect of CBD was accompanied by increased mPFC serotonin turnover. Together, these results indicate that female rats are susceptible to the biphasic effects of low THC doses on anxiety. These effects could depend on mPFC and NAc dopaminergic and serotoninergic neurotransmissions. CBD could minimize potential THC high-dose side effects whereas enhancing the anxiolytic action of its low doses in females.

Nucleus reuniens of the thalamus controls fear memory reconsolidation.

The nucleus reuniens has been shown to support the acquisition, consolidation, maintenance, destabilization upon retrieval, and extinction of aversive memories. However, the direct participation of this thalamic subregion in memory reconsolidation is yet to be examined. The present study addressed this question in contextually fear-conditioned rats. Post-reactivation infusion of the GABAA receptor agonist muscimol, the glutamate N2A-containing NMDA receptor antagonist TCN-201, or the protein synthesis inhibitor anisomycin into the NR induced significant impairments in memory reconsolidation. Administering muscimol or TCN-201 and anisomycin locally, or associating locally infused muscimol or TCN-201 with systemically administered clonidine, an α2-receptor adrenergic agonist that attenuates the noradrenergic tonus associated with memory reconsolidation, produced no further reduction in freezing times when compared with the muscimol-vehicle, TCN-201-vehicle, vehicle-anisomycin, and vehicle-clonidine groups. This pattern of results indicates that such treatment combinations produced no additive/synergistic effects on reconsolidation. It is plausible that NR inactivation and antagonism of glutamate N2A-containing NMDA receptors weakened/prevented the subsequent action of anisomycin and clonidine because they disrupted the early stages of signal transduction pathways involved in memory reconsolidation. It is noteworthy that these pharmacological interventions, either alone or combined, induced no contextual memory specificity changes, as assessed in a later test in a novel and unpaired context. Besides, omitting memory reactivation precluded the impairing effects of muscimol, TCN-201, anisomycin, and clonidine on reconsolidation. Together, the present findings demonstrate interacting mechanisms through which the NR can regulate contextual fear memory restabilization.

Taking advantage of fear generalization-associated destabilization to attenuate the underlying memory via reconsolidation intervention.

Upon retrieval, an aversive memory can undergo destabilization and reconsolidation. A traumatic-like memory, however, may be resistant to this process. The present study sought to contribute with a strategy to overcome this potential issue by investigating whether generalized fear retrieval is susceptible to destabilization-reconsolidation that can be pharmacologically modified. We hypothesized that exposure to a context that elicits moderate generalization levels would allow a malleable memory state. We developed a fear conditioning protocol in context A (cxt-A) paired with yohimbine administration to promote significant fear to a non-conditioned context B (cxt-B) in rats, mimicking the enhanced noradrenergic activity reported after traumatic events in humans. Next, we attempted to impair the reconsolidation phase by administering clonidine (CLO) immediately after exposure to cxt-A, cxt-B, or a third context C (cxt-C) neither conditioned nor generalized. CLO administered post-cxt-B exposure for two consecutive days subsequently resulted in decreased freezing levels in cxt-A. CLO after cxt-B only once, after cxt-A or cxt-C in two consecutive days, or independently of cxt-B exposures did not affect fear in a later test. A 6-h-delay in CLO treatment post-cxt-B exposures produced no effects, and nimodipine administered pre-cxt-B exposures precluded the CLO action. We then quantified the Egr1/Zif268 protein expression following cxt-B exposures and CLO treatments. We found that these factors interact to modulate this memory destabilization-reconsolidation mechanism in the basolateral amygdala but not the dorsal CA1 hippocampus. Altogether, memory destabilization can accompany generalized fear expression; thus, we may exploit it to potentiate reconsolidation blockers' action.

A single dose of the organophosphate triazophos induces fear extinction deficits accompanied by hippocampal acetylcholinesterase inhibition.

Acute organophosphate (OP) poisoning, particularly by suicide attempts, generates high mortality and morbidity. Few studies have systematically addressed the consequences of acute OP intoxication on cognition and memory of survivors. Preclinical evidence suggests that acute OP-induced effects are associated with inhibiting the brain acetylcholinesterase (AChE) enzyme. The OP triazophos has been used worldwide, although its effects on mnemonic processing are yet to be investigated. Based on the above, the present study investigated whether acute triazophos intoxication interferes with the expression and extinction of contextual fear memory in rats. Hippocampal and amygdalar AChE activity and plasma butyrylcholinesterase (BChE) were measured at the end of the experiment to confirm the cholinergic overstimulation. Independent cohorts of animals intoxicated with triazophos were evaluated in the novel object recognition (NOR) test, a less aversive associative memory task. At the dose of 15 mg/kg, triazophos administered immediately after contextual fear conditioning impaired the extinction but not the expression of freezing behavior. Triazophos poisoning induced no changes in the discrimination index in the NOR test. Triazophos inhibited the AChE activity in a time- and brain region-dependent manner. Our findings suggest that fear memory extinction deficits induced by acute triazophos intoxication are accompanied by hippocampal AChE inhibition. The deficient fear extinction associated with acute OP poisoning may represent a behavioral and biochemical phenotype helpful to study mechanisms of neurotoxicity and treatment approach of OP suicide survivors.

Thalamic nucleus reuniens regulates fear memory destabilization upon retrieval.

The neural circuit supporting aversive memory destabilization after retrieval includes the hippocampus, amygdala, and medial prefrontal cortex. The nucleus reuniens (NR) contributes to the functional interaction of these brain regions relevant to cognitive processing. However, the direct participation of this thalamic subregion in memory destabilization is yet to be investigated. The present study addressed this question in contextually fear-conditioned rats. Pre-reactivation infusion of the GABAA receptor agonist muscimol, the protein degradation inhibitor clasto-lactacystin ß-lactone (ß-lac), or the glutamate N2B-containing NMDA receptors antagonist ifenprodil into the NR prevented the post-reactivation amnestic effects of both locally infused anisomycin and systemically administered clonidine. In either case, the results suggest a significant disruption in memory destabilization. It is noteworthy that these pharmacological interventions induced no changes in expression or contextual specificity of the memory. Moreover, omitting memory reactivation precluded the muscimol, ß-lac, and ifenprodil effects on destabilization and the anisomycin and clonidine effects on reconsolidation. We also quantified the Egr1/Zif268-expressing neurons to investigate the effects of muscimol-induced NR inactivation on the activity-related plasticity locally, and in other brain regions supporting fear memory destabilization-reconsolidation. Relative to controls, there were reduced values in the NR, the dorsal CA1 hippocampus, the prelimbic cortex, and the infralimbic cortex. In contrast, increases happened in the ventral CA1 hippocampus and the basolateral amygdala. These results suggest that NR has a circuit-level influence on this process. Together, present findings demonstrate how the NR can regulate contextual fear memory destabilization upon retrieval.

Infralimbic cortex controls fear memory generalization and susceptibility to extinction during consolidation.

Lesioning or inactivating the infralimbic (IL) subregion of the medial prefrontal cortex before acquisition produces more generalized and extinction-resistant fear memories. However, whether and how it modulates memory specificity and extinction susceptibility while consolidation takes place is still unknown. The present study aims to investigate these questions using muscimol-induced temporary inactivation and anisomycin-induced protein synthesis inhibition in the rat IL following contextual fear conditioning. Results indicate that the IL activity immediately after acquisition, but not six hours later, controls memory generalization over a week, regardless of its strength. Such IL function depends on the context-shock pairing since muscimol induced no changes in animals exposed to immediate shocks or the conditioning context only. Animals in which the IL was inactivated during consolidation extinguished similarly to controls within the session but were unable to recall the extinction memory the following day. Noteworthy, these post-acquisition IL inactivation-induced effects were not associated with changes in anxiety, as assessed in the elevated plus-maze test. Anisomycin results indicate that the IL protein synthesis during consolidation contributes more to producing extinction-sensitive fear memories than memory specificity. Collectively, present results provide evidence for the IL's role in controlling generalization and susceptibility to extinction during fear memory consolidation.

Role of prelimbic cortex PKC and PKMζ in fear memory reconsolidation and persistence following reactivation.

The persistence of newly acquired memories is supported by the activity of PKMζ, an atypical isoform of protein kinase C (PKC). Whether the activity of conventional and atypical PKC isoforms contributes to reactivated memories to persist is still unknown. Similarly, whether memory reactivation is a prerequisite for interventions to be able to change memory persistence is scarcely investigated. Based on the above, we examined the role of conventional and atypical PKC isoforms in the prelimbic cortex in reconsolidation and persistence of a reactivated contextual fear memory in male Wistar rats. It is shown that (i) inhibiting the PKC activity with chelerythrine or the PKMζ activity with ZIP impaired the persistence of a reactivated memory for at least 21 days; (ii) ZIP given immediately after memory reactivation affected neither the reconsolidation nor the persistence process. In contrast, when given 1 h later, it impaired the memory persistence; (iii) chelerythrine given immediately after memory reactivation impaired the reconsolidation; (iv) omitting memory reactivation prevented the chelerythrine- and ZIP-induced effects: (v) the ZIP action is independent of the time elapsed between its administration and the initial memory test. The results indicate that prelimbic cortex PKC and PKMζ are involved in memory reconsolidation and persistence.

Role of dorsal hippocampus κ opioid receptors in contextual aversive memory consolidation in rats.

The main κ opioid receptors (κORs) subtypes already described (κ1ORs and κ2ORs) are expressed in brain regions involved in aversive memory consolidation, including the dorsal hippocampus (DH). However, the role of DH κORs in consolidation of aversive memories with varied intensity and specificity is still uncertain. The present study aimed to investigate this question using pharmacological agents in rats subjected to a weak, moderate or strong contextual aversive conditioning (CAC) protocol. Antagonizing DH κORs with nor-binaltorphimine (nor-BNI), immediately after, but not 6 h later, a moderate CAC leads to intensified freezing behavior in the re-exposure to the paired context. Thus, indicating that DH κORs have an inhibitory role in the consolidation of an aversive memory. Increased DH κORs expression 1 h and 3 h after the moderate CAC was also observed. This up-regulation was absent in animals only exposed to the shock or to the context, indicating that this phenomenon requires a shock-context pairing to occur. Intra-DH nor-BNI infusion induced no changes following a weak CAC, but it was able to potentiate the expression of freezing behavior in novel and unpaired context after a strong CAC, indicating that DH κORs also modulate the consolidation of a more intense and generalized memory. Moreover, infusing the κ2ORs agonist GR 89696, but not the κ1ORs agonist U-69593, into the DH reduced the conditioned freezing expression. Nor-BNI pretreatment in a sub-effective dose prevented the κ2ORs agonist effects. Altogether, the present findings provide convergent evidence that κORs activation negatively modulates contextual aversive memory consolidation in rat dorsal hippocampus.

Anandamide reverses depressive-like behavior, neurochemical abnormalities and oxidative-stress parameters in streptozotocin-diabetic rats: Role of CB1 receptors.

The pathophysiology associated with increased prevalence of depression in diabetics is not completely understood, although studies have pointed the endocannabinoid system as a possible target. Then, we aimed to investigate the role of this system in the pathophysiology of depression associated with diabetes. For this, diabetic (DBT) male Wistar rats were intraperitoneally treated with cannabinoid CB1 (AM251, 1mg/kg) or CB2 (AM630, 1mg/kg) receptor antagonists followed by anandamide (AEA, 0.005mg/kg) and then submitted to the forced swimming test (FST). Oxidative stress parameters, CB1 receptor expression and serotonin (5-HT) and noradrenaline levels in the hippocampus (HIP) and prefrontal cortex (PFC) were also performed. It was observed that DBT animals presented a more pronounced depressive-like behavior and increase of CB1 receptor expression in the HIP. AEA treatment induced a significant improvement in the depressive-like behavior, which was reversed by the CB1 antagonist AM251, without affecting the hyperglycemia or weight gain. AEA was also able to restore the elevated CB1 expression and also to elevate the reduced level of 5-HT in the HIP from DBT animals. In addition, AEA restored the elevated noradrenaline levels in the PFC and induced a neuroprotective effect by restoring the decreased reduced glutathione and increased lipid hydroperoxides levels along with the decreased superoxide dismutase activity observed in HIP or PFC. Together, our data suggest that in depression associated with diabetes, the endocannabinoid anandamide has a potential to induce neuroadaptative changes able to improve the depressive-like response by its action as a CB1 receptor agonist.

Neuroanatomy of anxiety.

The evolutionary approach to human anxiety is based on the defensive responses that nonhuman animals show to fear-provoking stimuli. Studies performed mostly on rodents have related areas such as the medial prefrontal cortex, amygdaloid and hypothalamic nuclei, hipoccampal formation, and midbrain central gray to these responses. It is clear, however, that animals show different and sometimes opposite responses according to the threatening stimulus. These responses include immediate reactions such as freezing or flight, behavioral inhibition or avoidance, which are organized by at least partially distinct brain systems. As discussed in this chapter, several pieces of evidence indicate that these brain systems are similar in rodents and primates. In addition, recent neuroimaging studies also suggest dysfunctions in these systems are probably related to anxiety disorders in humans.

Cholecystokinin-2 receptors modulate freezing and escape behaviors evoked by the electrical stimulation of the rat dorsolateral periaqueductal gray.

Systemic injection of the cholecystokinin type 2 (CCK(2)) receptor agonist CCK-4 evokes panic attacks in humans and facilitates the expression of a panic-related defensive behavior, escape, in rats. Given the prominent role attributed to the dorsal periaqueductal gray (dPAG) in the pathophysiology of panic, this midbrain area has been assumed to be one of the key regions mediating these effects of CCK-4. However, only a few studies have directly investigated the role of dPAG CCK(2) receptors in the regulation of panic-related behaviors. Even more disappointingly, the results of these investigations have been far from conclusive. In the present study we further addressed this issue by evaluating the effect of the intra-dorsolateral periaqueductal gray (dlPAG) injection of CCK-4 on two panic-related defensive behaviors, freezing and escape, evoked in male Wistar rats by the electrical stimulation of the dlPAG. The effects of CCK-4 (0.005-0.5 microg/0.2 microl) were compared to those caused by the local microinjection of the CCK(2) receptor antagonist LY225910 (0.001-1.0 microg/0.2 microl). The results showed that whereas CCK-4 facilitated the expression of both freezing and escape behaviors, LY225910 had the opposite effect. Pretreatment with an ineffective dose of LY225910 prevented the panicogenic-like effect of CCK-4. These results strengthen the view that CCK(2) receptors located in the dlPAG are involved in the regulation of panic-related behaviors and may mediate the effect of CCK-4 on panic.

Involvement of dorsolateral periaqueductal gray N-methyl-D-aspartic acid glutamate receptors in the regulation of risk assessment and inhibitory avoidance behaviors in the rat elevated T-maze.

The involvement of the dorsolateral periaqueductal gray in the regulation of fear-related behaviors such as escape and freezing is well established. It is still a matter of investigation, however, whether this midbrain area may have a relevant role in the modulation of more subtle defensive responses associated with anxiety such as risk assessment and inhibitory avoidance. By stimulating N-methyl-D-aspartic acid glutamate receptors located in the dorsolateral periaqueductal gray with its prototypical agonist N-methyl-D-aspartic acid (50 pmol), we report here an increase in both risk assessment and inhibitory avoidance behaviors of male Wistar rats tested in the elevated T-maze. These results are indicative of an anxiogenic-like effect. The selective N-methyl-D-aspartic acid receptor antagonist DL-2-amino-7-phosphonoheptanoic acid (2.0 and 4.0 nmol) had the opposite effect on both defensive tasks. Pretreatment with an ineffective dose of DL-2-amino-7-phosphonoheptanoic acid (1.0 nmol) prevented the N-methyl-D-aspartic acid anxiogenic-like effect. At the dose range of DL-2-amino-7-phosphonoheptanoic acid and/or N-methyl-D-aspartic acid tested, neither the escape response from one of the elevated T-maze open arms nor the general exploratory activity as assessed in the open-field test was affected. The present results suggest that the dorsolateral periaqueductal gray column is also involved in the regulation of defensive behaviors related to anxiety, and N-methyl-D-aspartic acid glutamate receptors are recruited for this action.

Further evidence that anxiety and memory are regionally dissociated within the hippocampus.

The hippocampus has been implicated in the regulation of anxiety and memory processes. Nevertheless, the precise contribution of its ventral (VH) and dorsal (DH) division in these issues still remains a matter of debate. The Trial 1/2 protocol in the elevated plus-maze (EPM) is a suitable approach to assess features associated with anxiety and memory. Information about the spatial environment on initial (Trial 1) exploration leads to a subsequent increase in open-arm avoidance during retesting (Trial 2). The objective of the present study was to investigate whether transient VH or DH deactivation by lidocaine microinfusion would differently interfere with the performance of EPM-naive and EPM-experienced rats. Male Wistar rats were bilaterally-implanted with guide cannulas aimed at the VH or the DH. One-week after surgery, they received vehicle or lidocaine 2.0% in 1.0 microL (0.5 microL per side) at pre-Trial 1, post-Trial 1 or pre-Trial 2. There was an increase in open-arm exploration after the intra-VH lidocaine injection on Trial 1. Intra-DH pre-Trial 2 administration of lidocaine also reduced the open-arm avoidance. No significant changes were observed in enclosed-arm entries, an EPM index of general exploratory activity. The cautious exploration of potentially dangerous environment requires VH functional integrity, suggesting a specific role for this region in modulating anxiety-related behaviors. With regard to the DH, it may be preferentially involved in learning and memory since the acquired response of inhibitory avoidance was no longer observed when lidocaine was injected pre-Trial 2.

Involvement of dorsolateral periaqueductal gray cholecystokinin-2 receptors in the regulation of a panic-related behavior in rats.

Cholecystokinin (CCK) has been implicated in anxiety disorders. The midbrain periaqueductal gray (PAG), which modulates anxiety and panic reactions, contains CCK-immunoreactive fibers and CCK(2) receptors. The present study investigated the involvement of CCK(2) receptors of the PAG dorsolateral subdivision (dlPAG) in the regulation of inhibitory avoidance and escape, two defensive behaviors that have been related in terms of psychopathology to generalized-anxiety and panic disorders, respectively. Male Wistar rats were microinjected in the dlPAG with the CCK(2) receptor agonist cholecystokinin-tetrapeptide (CCK-4; 0.08-0.32 nmol/0.2 microL), the CCK(2) receptor antagonist LY-225910 (0.05-0.20 nmol/0.2 microL) or LY-225910 prior to CCK-4. Inhibitory avoidance and escape behaviors were evaluated in the elevated T-maze. Whereas CCK-4 facilitated escape, indicating a panic-like action, LY-225910 had the opposite effect. Pretreatment with a non-effective dose of LY-225910 prevented the panic-eliciting action of CCK-4. Neither CCK-4 nor LY-225910 affected inhibitory avoidance acquisition. The present results substantiate the view that dlPAG CCK(2) receptors modulate panic-related behaviors.

Enhanced dorsolateral periaqueductal gray activity counteracts the anxiolytic response to midazolam on the elevated plus-maze Trial 2 in rats.

Rodents previously (Trial 1) experienced in the elevated plus-maze (EPM) apparatus no longer respond to anxiolytic-like drugs during retesting (Trial 2). In view of the fact that the dorsolateral periaqueductal gray (dlPAG) modulates fear/anxiety-like behavior, the present study sought to determine its role in this phenomenon. In order to address this issue, EPM-experienced rats that had received lidocaine, a drug which produces a reversible functional deactivation, intra-dlPAG pre-Trial 1, post-Trial 1 or pre-Trial 2, were systemically injected with the benzodiazepine midazolam and submitted to the EPM apparatus. According to the results, 0.25 mg/kg midazolam increased open arms exploration and reduced risk assessment behavior, suggesting an anxiolytic-like effect in EPM-naive rats, regardless of the intra-dlPAG treatment. EPM-experienced rats administered with midazolam only displayed a similar pattern of behavior when lidocaine was administered intra-dlPAG pre-Trial 2, but not pre- or post-Trial 1. These effects were observed in the absence of changes in enclosed arms entries, an EPM general exploratory activity index. The present results suggest that an increased activity of the dlPAG during Trial 2 would explain the lack of anxiolytic-like effect of drugs elicited by prior EPM test experience.