Conditioned Emotional Response Test Research Articles

Reply to the comment on: ‘Anxiogenic-like effects of chronic cannabidiol administration in rats’ (Elbatsh MM, Assareh N, Marsden CA and Kendall DA; Psychopharmacology 2012)

In reply to Dr. Gururajan's comments on our manuscript (Elbatsh et al. 2012) about the anxiogenic-like effects of chronic cannabidiol administration in rats, we accept that the use of a wider ranging dose/response design would have been desirable. We emphasised the need for additional longterm multi-dose studies of the drug in models of affective disease in the discussion which present constraints of finance and time have not allowed. Moreover, given that cannabidiol (CBD) is being reported almost as a panacea, we consider it of value to show, even if only at a single dose, that CBD can provoke potentially deleterious behavioural responses. Secondly, in terms of the behavioural changes in the context of anxiety, not only total distance travelled was reported in the activity boxes, but also rearing and grooming as possible markers of anxiety-related behaviour. Locomotor activity in a neutral environment was measured to detect any hypoactivity associated with CBD treatment that could have interfered with the interpretation of behavioural changes in the conditioned emotional response (CER). We used the CER test which had previously been used to show anxiolytic effects of acute CBD (Bitencourt et al. 2008; Resstel et al. 2006), but we agree that the elevated plus maze could also be an appropriate test, given that it would be useful to find converging effects in a number of anxiety test procedures. Maha Elbatsh for the authors

Anxiogenic-like effects of chronic cannabidiol administration in rats

Several pre-clinical and human-based studies have shown that acutely administered cannabidiol (CBD) can produce anxiolytic-like effects The present study investigated the effects of chronic administration of CBD on rat behaviour and on the expression of brain proteins. Male Lister-hooded rats (150-200 g, n = 8 per group) received daily injections of CBD (10 mg/kg, i.p.) for 14 days. The rats were subjected to two behavioural tests: locomotor activity and conditioned emotional response (CER). The expression of brain-derived neurotrophic factor (BDNF), its receptor tyrosine kinase B (Trk B), extracellular signal-regulated kinases (ERK1/2) and phospho-ERK1/2 and the transcription factor cyclic AMP response element binding protein activation (CREB) and phospho-CREB were determined in brain regions such as the frontal cortex and hippocampus using Western immunoblotting. CBD significantly increased the time spent freezing in the CER test with no effect on locomotor activity. CBD significantly reduced BDNF expression in the hippocampus and frontal cortex with no change in the striatum. In addition, CBD significantly reduced TrkB expression in the hippocampus with a strong trend towards reduction in the striatum but had no effect in the frontal cortex. In the hippocampus, CBD had no effect on ERK1/2 or phospho-ERK2, but in the frontal cortex, CBD significantly reduced phospho-ERK1/2 expression without affecting total ERK. Chronic administration of CBD produced an anxiogenic-like effect in clear opposition to the acute anxiolytic profile previously reported. In addition, CBD decreased the expression of proteins that have been shown to be enhanced by chronic treatment with antidepressant/anxiolytic drugs.

Fluoxetine improves the memory deficits caused by the chemotherapy agent 5-fluorouracil

Cancer patients who have been treated with systemic adjuvant chemotherapy have described experiencing deteriorations in cognition. A widely used chemotherapeutic agent, 5-fluorouracil (5-FU), readily crosses the blood–brain barrier and so could have a direct effect on brain function. In particular this anti mitotic drug could reduce cell proliferation in the neurogenic regions of the adult brain. In contrast reports indicate that hippocampal dependent neurogenesis and cognition are enhanced by the SSRI antidepressant Fluoxetine. In this investigation the behavioural effects of chronic (two week) treatment with 5-FU and (three weeks) with Fluoxetine either separately or in combination with 5-FU were tested on adult Lister hooded rats. Behavioural effects were tested using a context dependent conditioned emotional response test (CER) which showed that animals treated with 5-FU had a significant reduction in freezing time compared to controls. A separate group of animals was tested using a hippocampal dependent spatial working memory test, the object location recognition test (OLR). Animals treated only with 5-FU showed significant deficits in their ability to carry out the OLR task but co administration of Fluoxetine improved their performance. 5-FU chemotherapy caused a significant reduction in the number of proliferating cells in the sub granular zone of the dentate gyrus compared to controls. This reduction was eliminated when Fluoxetine was co administered with 5-FU. Fluoxetine on its own had no effect on proliferating cell number or behaviour. These findings suggest that 5-FU can negatively affect both cell proliferation and hippocampal dependent working memory and that these deficits can be reversed by the simultaneous administration of the antidepressant Fluoxetine.

The anti-epileptic drug lacosamide (Vimpat ®) has anxiolytic property in rodents

Lacosamide ((R)-2-acetamido-N-benzyl-3-methoxypropionamide; formerly harkoseride, SPM 927; Vimpat ®), has been recently approved by US and European regulatory authorities for use as add-on therapy for partial-onset seizures in adults. Because a number of anti-epileptic drugs are used to treat conditions beyond epilepsy, including anxiety, in the present study we investigated the anxiolytic potential of lacosamide in a conditioned emotional response (CER) model in rat, and the mouse marble burying assay. In each test lacosamide produced a significant effect consistent with anxiolysis, i.e. lacosamide increased suppression ratio in the CER test, and reduced the number of marbles buried in the marble burying assay. The doses necessary for an anxiolytic effect were higher than those necessary for efficacy in seizure tests conducted in the same species. For example in the mouse, the lacosamide oral ED 50 in the maximal electroshock seizure (MES) and 6 Hz tests was 5.3 and 9.6 mg/kg respectively, and the minimal effective dose in the marble burying assay was 30 mg/kg. In both seizure and anxiety tests, the (S)-enantiomer of lacosamide was inactive suggesting a similar mechanism of action, possibly use-dependent inhibition of sodium channel function (Errington et al., 2008). Efficacy in the CER model was equivalent to diazepam and pregabalin (Lyrica ®). In tests of side-effects, lacosamide had no effect on choice accuracy in the delayed match to position task of working memory, although at the 30 mg/kg dose, response rates and response latencies were significantly affected. In sum, the present results identify for the first time, an anxiolytic potential of lacosamide.

Nesfatin-1 increases anxiety- and fear-related behaviors in the rat

Nesfatin-1, derived from the protein NEFA/nucleobindin2 (NUCB2), is a newly identified peptide that acts as a potent satiety agent. It has been reported that peptides involved in the regulation of ingestive behavior are also involved in the regulation of the stress response. However, the relation between nesfatin-1 and stressor-related behaviors like anxiety and/or fear has not yet been investigated. The effects of intracerebroventricular (ICV) injection of nesfatin-1 (0, 5, and 25 pmol/3 microl) were assessed in several paradigms that are thought to reflect anxiety and/or fear in rats. Consistent with an anxiogenic effect, nesfatin-1 dose-dependently decreased the percentage of time spent on the open arms of the elevated plus maze, increased latency to approach, and decreased consumption of a palatable snack in an anxiogenic (unfamiliar) environment. Moreover, ICV nesfatin-1 increased the fear-potentiated startle response and the time spent freezing to both context and conditioned cues in a conditioned emotional response test. These findings suggest that in addition to its role as a satiety peptide, nesfatin-1 may also be involved in the mediation of anxiety- and/or fear-related responses.

Comparative effects of nonselective and subtype-selective gamma-aminobutyric acidA receptor positive modulators in the rat-conditioned emotional response test

Benzodiazepine receptor anxiolytics show no selectivity between gamma-aminobutyric acid-A receptors containing alpha1, alpha2, alpha3 or alpha5 subunits. Pharmacological studies and data emerging from transgenic mouse models, however, predict that compounds with selective affinity and/or efficacy for gamma-aminobutyric acid-A receptor subtypes would have novel pharmacological profiles. Thus, the gamma-aminobutyric acid-A-alpha1 'affinity selective' drug zolpidem has a sedative-hypnotic profile, whereas L838,417, which has 'selective efficacy' for gamma-aminobutyric acid-A-alpha2, alpha3 and alpha5 receptors, has an anxiolytic-like profile. Here, we compare the nonselective benzodiazepine-site-positive modulators diazepam, lorazepam, midazolam, alprazolam and zopiclone with (i) gamma-aminobutyric acid-AA-alpha1 affinity selective compounds zolpidem and CL218,872 and (ii) L838,417, in the rat-conditioned emotional response test after systemic administration. Given the role of the basolateral amygdala in anxiety and the expression of alpha1, alpha2 and alpha3 subunits in this region, we also assessed the effects of bilateral infusion of L838,417 and midazolam directly into basolateral amygdala in the conditioned emotional response test. Nonselective modulators at low-moderate doses produced anxiolytic effects and sedation at higher doses. Zolpidem was inactive as an anxiolytic and engendered severe sedation, whereas CL218,872 produced an anxiolytic-like profile with minimal sedation. L838,417 produced an anxiolytic-like profile with no sedation, albeit producing behavioural disturbance at high doses. Infusion of midazolam and L838,417 into basolateral amygdala engendered anxiolytic-like effects, although both compounds were more effective after systemic injections, implicating additional brain sites in their anxiolytic-like actions after systemic administration. In conclusion, the diversity of effects of the compounds studied implicates both intrinsic efficacy and/or subtype selectivity as important determinants of anxiolytic-like effects in the rat-conditioned emotional response test.

7-OH-DPAT effects on latent inhibition: low dose facilitation but high dose blockade: Implications for dopamine receptor involvement in attentional processes

7-OH-DPAT is a dopamine D2/D3 agonist, which at low doses acts preferentially on D3 receptors but at high doses it acts on D2 and D3 receptors. The present study investigated the contribution of D3 and D2 receptors on latent inhibition (LI) by using two dose levels of 7-OH-DPAT: a low dose, 0.1 mg/kg (D3 receptor activation) and a high dose, 1.0 mg/kg, (D2/D3 receptor activation) in a conditioned emotional response (CER) paradigm. The LI Protocols included CS pre-exposure (10 or 40 CS alone trials), CER induction and a non-drug CER test phase. Additionally, the drug effects upon CER acquisition without LI were assessed using the same treatments and test environment pre-exposure protocols but without the tone CS. The effects of 7-OH-DPAT on crossing, rearing and grooming were also measured in an open field 1 day after the CER test phase. The results showed that the low dose 7-OH-DPAT treatment potentiated LI at 10 but not at 40 CS pre-exposures. The high dose 7-OH-DPAT treatment blocked LI at both the 10 and 40 stimulus pre-exposures; and it also induced hyperactivity. Thus, D3 stimulation induced by a low dose of 7-OH-DPAT can facilitate LI but these effects are contingent upon and are specific to the number of stimulus presentations. Altogether, these findings indicate that D3 stimulation can enhance attentional processes, but D2 stimulation can impair attentional processes.

Both α2 and α3 GABAA receptor subtypes mediate the anxiolytic properties of benzodiazepine site ligands in the conditioned emotional response paradigm

Mice with point-mutated alpha2 GABAA receptor subunits (rendering them diazepam insensitive) are resistant to the anxiolytic-like effects of benzodiazepines (BZs) in unconditioned models of anxiety. We investigated the role of the alpha2 GABAA subtype in a model of conditioned anxiety. alpha2(H101R) and wildtype mice were trained in a conditioned emotional response (CER) task, in which lever-pressing for food on a variable interval (VI) schedule was suppressed during the presentation of a conditioned stimulus (CS+) that predicted footshock. The ability of diazepam, ethanol and pentobarbital to reduce suppression during the CS+ was interpreted as an anxiolytic response. Diazepam (0, 0.5, 1, 2, 4 and 8 mg/kg) induced a dose-dependent anxiolytic-like effect in wildtype mice. At high doses, diazepam (2, 4 and 8 mg/kg) was sedative in alpha2(H101R) mice. Analysis of the anxiolytic properties of nonsedative diazepam doses (0.5 and 1 mg/kg), showed that alpha2(H101R) mice were resistant to the anxiolytic effects of diazepam. Equivalent anxiolytic properties of pentobarbital (20 mg/kg) and ethanol (1 and 2 g/kg) were seen in both genotypes. These findings confirm the critical importance of the alpha2 GABAA subtype in mediating BZ anxiolysis. However, as a compound, L-838417, with agonist properties at alpha2, alpha3 and alpha5-containing receptors, gave rise to anxiolytic-like activity in alpha2(H101R) mice in the CER test, alpha3-containing GABA receptors are also likely to contribute to anxiolysis. Observations that alpha2(H101R) mice were more active, and displayed a greater suppression of lever pressing in response to fear-conditioned stimuli than wildtype mice, suggests that the alpha2(H101R) mutation may not be behaviourally silent.

972 ORAL Ifosfamide vs cyclophosphamide: long term gonadal effects in 116 male survivors of childhood cancer

The role of hippocampus in the anxiolytic-like effect of buspirone in the conditioned emotional response test (CER, a freezing response), was examined by immunocytochemical detection of the c-Fos protein. It was shown that buspirone at the dose of 0.5 and 1.5 mg/kg i.p. given before test session, which was 24 h after the aversive training, significantly decreased freezing response within a limited dose range of the U-shaped dose–response relationship. Exposure of animals to aversively conditioned context (a contextual fear) induced the production of c-Fos protein in the dentate gyrus, CA-1 and CA-3 layers of the hippocampus. Pretreatment with buspirone (1.5 mg/kg) significantly attenuated the effects of aversive memory on c-Fos protein expression in the CA-1 and CA-3 layers of the hippocampus. These immunocytochemical results support previous data obtained in our laboratory with the help of selective neurotoxic lesions and intrahippocampal drug injections suggesting an important role of hippocampus in the anxiolytic effects of buspirone.

Buspirone attenuates conditioned fear-induced c-Fos expression in the rat hippocampus

The role of hippocampus in the anxiolytic-like effect of buspirone in the conditioned emotional response test (CER, a freezing response), was examined by immunocytochemical detection of the c-Fos protein. It was shown that buspirone at the dose of 0.5 and 1.5 mg/kg i.p. given before test session, which was 24 h after the aversive training, significantly decreased freezing response within a limited dose range of the U-shaped dose–response relationship. Exposure of animals to aversively conditioned context (a contextual fear) induced the production of c-Fos protein in the dentate gyrus, CA-1 and CA-3 layers of the hippocampus. Pretreatment with buspirone (1.5 mg/kg) significantly attenuated the effects of aversive memory on c-Fos protein expression in the CA-1 and CA-3 layers of the hippocampus. These immunocytochemical results support previous data obtained in our laboratory with the help of selective neurotoxic lesions and intrahippocampal drug injections suggesting an important role of hippocampus in the anxiolytic effects of buspirone.

Lamotrigine has an anxiolytic-like profile in the rat conditioned emotional response test of anxiety: a potential role for sodium channels?

Many anticonvulsants are used in disorders other than epilepsy. For example, lamotrigine is reported to be effective in post-traumatic stress disorder and mania. We assessed the effects of the anticonvulsants lamotrigine, valproate and carbamazepine in an animal model of anxiety. We assessed a wide range of pharmacological tools to delineate the mechanism of lamotrigine's anxiolytic effect. We assessed these compounds in the rat conditioned emotional response (CER) test of anxiety. Lamotrigine (30-80 mg/kg) dose-dependently and reproducibly engendered an anxiolytic response in this test, with similar efficacy to benzodiazepines. Carbamazepine (20-40 mg/kg) and riluzole (10 mg/kg), which block Na+ channels by a similar mechanism as lamotrigine, were also anxiolytic. By contrast, valproate (100-600 mg/kg) was inactive and appears to differ in its interaction with Na+ channels. The SSRI paroxetine, the GABA(A) receptor positive modulator propofol, the NMDA antagonists memantine and (+)MK-801, and the Ca2+ channel antagonist nifedipine were all inactive in the CER test, suggesting these mechanisms may not mediate the anxiolytic effect of lamotrigine. More directly, we showed that the anxiolytic effect of lamotrigine could be blocked by co-administering rats with the Na+ channel activator veratrine (0.1 mg/kg). By contrast, neither the Ca2+ channel agonist BAYK8644 (0.5 mg/kg) nor the 5-HT1A or 5-HT(1/2) antagonists WAY100635 (0.3 mg/kg) and metergoline (3 mg/kg), respectively, were able to block the effect. Lamotrigine's anxiolytic effect in the CER test may be mediated via block of Na+ channels, and this may represent a target for the development of novel anxiolytics.

Effects of 5-HT1A receptor agonists, partial agonists and a silent antagonist on the performance of the conditioned emotional response test in the rat.

In the present study, the effects of 5-HT1A receptor ligands with varying degrees of intrinsic activity at the 5-HT1A receptor were examined in the conditioned emotional response (CER) test and their effects compared to those of the benzodiazepine receptor agonists, diazepam and chlordiazepoxide. Diazepam (3.0 mg/kg) and chlordiazepoxide (3.0 mg/kg), and the 5-HT1A receptor partial agonists, ipsapirone (10.0 mg/kg) and gepirone (3.0 mg/kg), alleviated conditioned suppression of lever pressing. The 5-HT1A receptor partial agonist, buspirone (0.1-1.0 mg/kg), the 5-HT1A receptor agonist, 8-OH-DPAT (0.01-0.10 mg/kg), and the 5-HT1A receptor antagonist, WAY-100635 (0.03-3.0 mg/kg), had no effects on conditioned fear. Neither enhancing the level of food deprivation nor pretreatment with the amnesic agent scopolamine induced anxiolytic-like effects in the present CER test. The anxiolytic-like effects of ipsapirone in this test were completely reversed by WAY-100635. These results indicate that 5-HT1A agonist, but not antagonist actions, induce an anxiolytic effect in the CER test in rats.

Lack of effect of flumazenil and CGS 8216 on the anxiolytic-like properties of loreclezole

Loreclezole is a novel antiepileptic that interacts in a unique way with the GABA A receptor complex. Its anticonvulsant effect in rats is reversed by benzodiazepine receptor partial inverse agonists, such as CGS 8216 but not by the competitive benzodiazepine receptor antagonist, flumazenil (Ro 15-1788). In this study loreclezole (30.0–50.0 mg/kg i.p.) was found to induce an anxiolytic-like effect in a rat conditioned emotional response test that was reversed neither by flumazenil (10.0 mg/kg) nor by CGS 8216 (2-phenylpyrazolo[4,3- c]quinolin-3(5 H)-one, 0.3–3.0 mg/kg). The results suggest that the anxiolytic-like effects of loreclezole are not mediated by the benzodiazepine receptor.

Restoration of shock‐suppressed behavior by treatment with (+)‐5‐methyl‐10,11‐dihydro‐5H‐dibenzo[a, d]cyclohepten‐5, 10‐imine (MK‐801), a substance with potent anticonvulsant, central sympathomimetic, and apparent anxiolytic properties

AbstractMK‐801 was evaluated in rats for “antipunishment” and “anticonflict” activity in two procedures: (1) A conditioned emotional response (CER) test involving the suppression of lever‐pressing by unaviodable shock and (2) a simple conflict test in water‐deprived animals that were shocked for licking water. The effect of MK‐801 in both procedures was qualitatively similar to the benzodiazepines. Lever‐pressing in the CER test was increased by MK‐801 at doses ranging from 50–400 μg/kg administered orally (p.o.) at either 0.5 or 2 hours prior to testing. The number of shocks received in the “thirsty rat” conflict procedure was increased by MK‐801 at doses from 110–1,000 μg/kg p.o., providing the compound was given 2 or more hours before test. MK‐801 was without anticonflict activity when administered 1 hour prior to study.In squirrel monkeys trained in a response‐contingent conflict paradigm, a specific anticonflict effect for MK‐801 (50–400 μ/kg p.o.) was not demonstrable. As assessed by observing the overt behavior of squirrel monkeys, MK‐801 at doses greater than 100 μg/kg p.o. caused apparent “taming” or “tranquilization.” Chlordizepoxide and diazepam given, respectively, at doses above 1 and 2 mg/kg p.o. had a similar “taming” action. The benzodiazepines possessed a greater separation between doses producing “taming” and those causing ataxia than did MK‐801.The mode of action for the antipunishment effect of MK‐801 in rats is not known, but it was found that naloxone (2 or 5 mg/kg SC) antagonized the anticonflict actions of both MK‐801 and chlordiazepoxide. In vitro, MK‐801 was inactive (IC50 > 2 μM) with respect to competing for binding to rat brain tissue by various radioligands (diazepam, muscimol, apomorphine, spiroperidol, serotonin, LSD, WB‐4101, dihydroalprenolol, QNB, and 2‐chloroadenosine). An increase in 3H‐diazepam binding in vitro in rat brain tissue was detected following acute, but not chronic, treatment in vivo with MK‐801.

Catecholaminergic drug and conditioned emotional response in the white rat

This experiment was conducted to examine the effect of change of catecholamine (CA) by catecholaminergic drug on animal emotional behavior. 32 male wister strain albino rats were randomly allocated to one of four groups. As summerized in Table 1, α-methyl-p-tyrosine (α-MT, 75 mg/kg, i. p.), namely CA synthesis inhibitor was administered 4.5hr. before test for Group A and Group AD. For Group D and Group AD, L-DOPA (50 or 100 mg/kg, i. p.), namely the precursor of CA was administered 1.5 hr. before test. For Group S, saline was administered. Animals were tested by conditioned emotional response (CER) in operant conditioning in Skinner box.L-DOPA significantly increased conditioned suppression in CER. α-MT alone did not affect conditioned suppression in CER, but pretreatment of α-MT significantly suppressed the increase of conditioned suppression by L-DOPA (Fig. 2, Fig. 3). According to the above results, it is supposed that increase of CA by L-DOPA enhanced emotionality and CER in Skinner box, whereas α-MT suppressed the enhancement of emotionality by L-DOPA. It was suggested that CER test measure is sensitive to slight increase of CA by low dose of L-DOPA.