A partial habituation method to test for anterograde and retrograde amnestic treatment effects: Evidence that antagonism of the NMDA receptor can induce anterograde but not retrograde amnestic effects.

BACKGROUND: A progressive decrease in spontaneous locomotion with repeated exposure to a novel environment has been assessed using both within and between-session measures. While both are well-established and reliable measurements, neither are useful alone as methods to concurrently assess treatment effects on acquisition and retention of habituation. NEW METHOD: We report a behavioral method that measures habituation by combining the within and between measurements of locomotion. We used a 30 min session divided into 6 five min blocks. In the first novel environment session activity was maximal in the first 5 min block but was reduced to a low level by the sixth block, indicative of within-session habituation. Using 8 daily sessions, we showed that this terminal block low level of activity progressed incrementally to the first block to achieve complete habituation. RESULTS/COMPARISON WITH EXISTING METHODS: Within-session activity across sessions was used to identify different stages of between session habituation. It was then possible to assess drug treatment effects from partial to complete habituation, so that treatment effects on retention of the previously acquired partial habituation, expressed as a reversion to an earlier within session habituation pattern (retrograde amnesia assessment), as well as the effects on new learning by the failure in subsequent sessions to acquire complete between-session habituation (anterograde amnesia assessment). CONCLUSIONS: The use of spontaneous motor activity to assess learning and memory effects provides the opportunity to assess direct treatment effects on behavior and motor activity in contrast to many learning and memory models.

The stingless bee Trigona spinipes (Hymenoptera: Apidae) is at risk from a range of insecticides via direct ingestion and trophallactic exchanges.

BACKGROUND: The stingless bee, Trigona spinipes, is an important pollinator of numerous native and cultivated plants. Trigona spinipes populations can be negatively impacted by insecticides commonly used for pest control in crops. However, this species has been neglected in toxicological studies. Here we observed the effects of seven insecticides on the survival of bees that had fed directly on insecticide-contaminated food sources or received insecticides via trophallactic exchanges between nestmates. The effects of insecticides on flight behavior were also determined for the compounds considered to be of low toxicity. RESULTS: Imidacloprid, spinosad and malathion were categorized as highly toxic to T. spinipes, whereas lambda-cyhalothrin, methomyl and chlorfenapyr were of medium to low toxicity and interfered with two aspects of flight behavior evaluated here. Chlorantraniliprole was the only insecticide tested here that had no significant effect on T. spinipes survival, although it did interfere with one aspect of flight capacity. A single bee that had ingested malathion, spinosad or imidacloprid, could contaminate three, four and nineteen other bees, respectively via trophallaxis, resulting in the death of the recipients. CONCLUSION: This is the first study to evaluate the ecotoxicology of a range of insecticides that not only negatively affected T. spinipes survival, but also interfered with flight capacity, a very important aspect of pollination behavior. The toxicity of the insecticides was observed following direct ingestion and also via trophallactic exchanges between nestmates, highlighting the possibility of lethal effects of these insecticides spreading throughout the colony, reducing the survival of non-foraging individuals. © 2023 Society of Chemical Industry.

The Potential of Metarhizium anisopliae Blastospores to Control Aedes aegypti Larvae in the Field.

Entomopathogenic fungi are promising as an environmentally benign alternative to chemical pesticides for mosquito control. The current study investigated the virulence of Metarhizium anisopliae blastospores against Aedes aegypti under both laboratory and field conditions. Virulence bioassays of conidia and blastospores were conducted in the laboratory, while field simulation bioassays were conducted under two conditions: totally shaded (TS) or partially shaded (PS). In the first bioassay (zero h), the larvae were added to the cups shortly after the preparation of the blastospores, and in the subsequent assays, larvae were added to the cups 3, 6, 9, and 12 days later. The survival of the larvae exposed to blastospores in the laboratory was zero on day two, as was the case for the larvae exposed to conidia on the sixth day. Under TS conditions, zero survival was seen on the third day of the bioassay. Under PS conditions, low survival rates were recorded on day 7. For the persistence bioassay under PS conditions, low survival rates were also observed. Metarhizium anisopliae blastospores were more virulent to Ae. aegypti larvae than conidia in the laboratory. Blastospores remained virulent under field simulation conditions. However, virulence rapidly declined from the third day of field bioassays. Formulating blastospores in vegetable oil could protect these propagules when applied under adverse conditions. This is the first time that blastospores have been tested against mosquito larvae under simulated field conditions, and the current study could be the basis for the development of a new biological control agent.

Morphine and dopamine: Low dose apomorphine can prevent both the induction and expression of morphine locomotor sensitization and conditioning.

The disinhibition of dopamine neurons in the VTA by morphine is considered an important contributor to the reward potency of morphine. In this report, three experiments were conducted in which a low dose of apomorphine (0.05 mg/kg) was used as a pretreatment to reduce dopamine activity. Locomotor hyperactivity was used as the behavioral response to morphine (10.0 mg/kg). In the first experiment, five treatments with morphine induced the development of locomotor and conditioned hyperactivity that were prevented by apomorphine given 10 min prior to morphine. Apomorphine before either vehicle or morphine induced equivalent reductions in locomotion. In the second experiment, the apomorphine pretreatment was initiated after induction of a conditioned hyperactivity and apomorphine prevented the expression of the conditioning. To assess the effects of apomorphine on VTA and the nucleus accumbens, ERK measurements were carried out after the induction of locomotor and conditioned hyperactivity. Increased ERK activation was found and these effects were prevented by the apomorphine in both experiments. A third experiment was conducted to assess the effects of acute morphine on ERK before locomotor stimulation was induced by morphine. Acute morphine did not increase locomotion, but a robust ERK response was produced indicating that the morphine-induced ERK activation was not secondary to locomotor stimulation. ERK activation was again prevented by the apomorphine pretreatment. We suggest that contiguity between the ongoing behavioral activity and the morphine activation of the dopamine reward system incentivizes and potentiates the ongoing behavior generating equivalent behavioral sensitization and conditioned effects.

Production of Escovopsis weberi (Ascomycota: Hypocreales) Mycelial Pellets and Their Effects on Leaf-Cutting Ant Fungal Gardens.

The maintenance of the symbiosis between leaf-cutting ants and their mutualistic fungus Leucoagaricus gongylophorus Singer (Moller) is vital for the survival of both species. The specialist fungal parasite Escovopsis weberi Muchovej & Della Lucia is a threat to this symbiosis, causing severe damage to the fungal garden. Mycelial pellets are resistant fungal structures that can be produced under laboratory conditions. These structures were studied for use in biological pest control, but the production of mycelial pellets has not previously been documented in Escovopsis. One of the aims of this study was to induce Escovopsis weberi to produce mycelial pellets and investigate the potential of these pellets for the control of leaf-cutting ants. We compared the pathogenicity of Escovopsis weberi mycelial pellets and conidia against mini-colonies of Acromyrmex subterraneus subterraneus Forel when applied in the form of baits. Worker ants were able to distinguish mycelial pellets from conidia, as baits with mycelial pellets were more attractive to workers than those with conidia, causing a greater negative impact on colony health. All types of baits containing Escovopsis weberi influenced the foraging activity but only treatments with viable fungal propagules resulted in an increase in the quantity of waste material, with a significant negative impact on the fungal garden biomass. The results provided novel information regarding Escovopsis recognition by worker ants and differences between conidia and mycelial pellet dynamics in leaf-cutting ant colonies, with new perspectives for the biological control of these important pests.

Lethal and sublethal effects of insecticides used in the management of Plutella xylostella (Lepidoptera: Plutellidae) on the predator Cycloneda sanguinea L. (Coleoptera: Coccinellidae).

BACKGROUND: The application of synthetic insecticides is the main strategy used to reduce the damage caused by the diamondback moth Plutella xylostella in commercial Brassica crops. However, incorrect insecticide use can cause biological and ecological disturbances in agroecosystems. Cycloneda sanguinea is a generalist voracious predator and is distributed widely in cultivated and noncultivated ecosystems. This study investigated the efficiency of four insecticides for the control of P. xylostella and the lethal and sublethal effects of these insecticides on C. sanguinea. RESULTS: Spinosad (92% mortality) and chlorfenapyr (76% mortality) were highly toxic to P. xylostela. However, chlorantraniliprole (10% mortality) and methomyl (no mortality) were ineffective against this pest. Chlorantraniliprole was the only insecticide that was highly toxic to C. sanguinea by contact (90% mortality), however, it was nontoxic following the ingestion of chlorantraniliprole-contaminated aphids. Interestingly, ingestion of prey contaminated with methomyl and chlorfenapyr was highly toxic (100% mortality) to C. sanguinea. Spinosad was nontoxic to C. sanguinea via exposure to contaminated surfaces and following ingestion of contaminated prey. However, direct contact of the insects with both methomyl and spinosad significantly affected C. sanguinea flight activity (vertical flight and free-fall flight), whereas chlorfenapyr impacted vertical flight only. CONCLUSION: These findings showed that chlorantraniliprole was not only ineffective for the control of P. xylostela, but was also highly toxic to C. sanguinea. The results indicated that spinosad was efficient against P. xylostela and was of low toxicity to C. sanguinea; however, the deleterious effects of this insecticide on flight behavior could result in reduced predatory efficiency. © 2022 Society of Chemical Industry.

Context evoked morphine conditioned effects can be equivalent to morphine induced drug effects in terms of behavioral response and ERK activation in reward associated subcortical brain structures.

Conditioned drug cues can evoke brief drug-like responses. In this report we show that using brief test sessions, contextual cues can induce conditioned hyperlocomotion and ERK responses equivalent to morphine induced responses. To assess acute unconditioned effects, rats that received morphine (MOR-1) or vehicle (VEH-1) were immediately placed onto an arena for a 5-min locomotion recording session after which ERK was measured in the ventral tegmental area (VTA) and nucleus accumbens (NAc). There were no differences in locomotion between the groups. However, the MOR-1 group had strong ERK activation in VTA and NAc. To assess MOR-conditioned effects, a chronic phase was carried out according to a Pavlovian conditioning protocol. There were two MOR paired groups (MORP), one MOR unpaired (MOR-UP) group and two VEH groups. The treatments were administered over 5 daily five minute test sessions. The final conditioning test was on day 6, in which one of the MOR-P groups and one of the VEH groups received VEH (MOR-P/VEH-6 and VEH/VEH-6, respectively). The other MOR-P group and VEH group received MOR (MOR-P/MOR; VEH/MOR-6, respectively). The MOR-UP group received VEH (MOR-UP/VEH-6). Rats received the treatments immediately prior to a 5-minute arena test, and after the session ERK was measured. No morphine induced locomotor stimulation was observed on day 1 but on days 2 to 5, hyperlocomotion in both MOR-P groups occurred. On test day 6, the MOR-P/VEH-6 and the MOR-P/MOR-6 groups had comparable locomotor stimulant responses and similar ERK activity in the VTA and NAc. The MOR-UP group did not differ from the VEH group. We suggest that ERK activation evoked by acute morphine served as a Pavlovian unconditioned stimulus to enable the contextual cues to acquire morphine conditioned stimulus properties and increase the incentive value of the contextual cues.

A new method to study learning and memory using spontaneous locomotor activity in an open-field arena.

BACKGROUND: Reduced locomotion with repeated exposure to a novel environment is often used as a measure of the basic adaptive learning process of habituation. While this is a well-established and reliable measure of habituation, it is not useful for the investigation of neurobiological changes before and after habituation because of the uncontrolled differential activity levels in a novel versus habituated environment. In this study we report a behavioral method that uses spontaneous locomotion to measure habituation, in which the total spontaneous locomotion in an initially novel environment does not change with repeated testing but, the ratio of central to peripheral activity does change and is indicative of habituation. The test sessions are brief (5 min) and the locomotion is measured in 2 separate zones. The peripheral zone comprises 8/9 of the test arena and the central zone 1/9 of the arena. RESULTS/COMPARISON WITH EXISTING METHODS: In contrast to methods that use between-session reductions in locomotion to assess habituation, this method employs brief test sessions in which overall activity between sessions does not change, but the distribution of locomotion in the periphery versus the central zone of the arena does change. The brevity of the test session also enables us to utilize post-trial drug treatment protocols to impact memory consolidation. CONCLUSIONS: The progressive change in the central/peripheral activity ratio with repeated testing can be determined independently of total activity and provides a habituation acquisition function that permits the measurement of neurobiological changes without the complication of effects related to changes in locomotor activity per se. The present report also presents evidence that this method can be used with post-trial drug treatment protocols to study the learning and memory effects of the post-trial treatments without the use of explicit rewards and punishments.

Metarhizium anisopliae blastospores are highly virulent to adult Aedes aegypti, an important arbovirus vector.

BACKGROUND: The use of entomopathogenic fungi (EPF) for the control of adult mosquitoes is a promising alternative to synthetic insecticides. Previous studies have only evaluated conidiospores against adult mosquitoes. However, blastospores, which are highly virulent against mosquito larvae and pupae, could also be effective against adults. METHODS: Metarhizium anisopliae (ESALQ 818 and LEF 2000) blastospores and conidia were first tested against adult Aedes aegypti by spraying insects with spore suspensions. Blastospores were then tested using an indirect contact bioassay, exposing mosquitoes to fungus-impregnated cloths. Virulence when using blastospores suspended in 20% sunflower oil was also investigated. RESULTS: Female mosquitoes sprayed with blastospores or conidia at a concentration of 108 propagules ml-1 were highly susceptible to both types of spores, resulting in 100% mortality within 7 days. However, significant differences in virulence of the isolates and propagules became apparent at 107 spores ml-1, with ESALQ 818 blastospores being more virulent than LEF 2000 blastospores. ESALQ 818 blastospores were highly virulent when mosquitoes were exposed to black cotton cloths impregnated with blastospores shortly after preparing the suspensions, but virulence declined rapidly 12 h post-application. The addition of vegetable oil to blastospores helped maintain virulence for up to 48 h. CONCLUSION: The results showed that blastospores were more virulent to adult female Ae. aegypti than conidia when sprayed onto the insects or applied to black cloths. Vegetable oil helped maintain blastospore virulence. The results show that blastospores have potential for use in integrated vector management, although new formulations and drying techniques need to be investigated.

Morphine reward effects and morphine behavioral sensitization: The adventitious association of morphine activation of brain reward effects with ongoing spontaneous activity.

The development of sensitization is one of the hallmarks of addictive drugs such as morphine. We administered morphine (10 mg/kg; MOR) to induce locomotor sensitization and ERK activation in the VTA and NAc. In the first experiment, four groups of rats received five daily 30 min sessions in an open-field, and locomotion was measured. For the first four sessions, one group received MOR pre-test (MOR-P); a second group received vehicle pre-test (MOR-UP) and MOR 30 min post-test; the remaining 2 groups received vehicle (VEH) pre-test. On the fifth session, the MOR-P, MOR-UP, and one VEH group received MOR pre-test and the remaining VEH group received VEH. Sensitization emerged in the first 5 min and progressed over to the second and third 5 min blocks only in the MOR-P group. For the second experiment, 4 groups received MOR and 4 groups VEH, and were then returned to their home cage and after 5, 15, 30 or 60 min post-injection, were euthanized for ERK measurements in VTA and NAc. ERK activation increased and peaked at 5 min post injection in the MOR group and then declined to VEH levels by 30 min. Another two groups received either MOR or VEH immediately before a 5 min arena test and ERK was measured immediately post-test. MOR had no effect on locomotion but increased ERK in the VTA and NAc. The peak ERK activation in VTA reflected activation of reward systems by morphine that reinforced locomotor behavior and with repeated treatments, induced a sensitization effect.

Production of conidia using different culture media modifies the virulence of the entomopathogenic fungus Metarhiziumys against Aedes aegypti larvae.

BACKGROUND & OBJECTIVES: Entomopathogenic fungi are being investigated for the biological control of a range of mosquitoes. Metarhizium conidiospores (conidia) effectively kill Aedes aegypti larvae and could be deployed as an alternative to chemical insecticides. Conidial yield and virulence of fungi when cultured on three different types of solid media, was investigated. METHODS: Three culture media were tested: a) Sabouraud dextrose agar (SDA); b) rice flour yeast agar (RYA) and c) rice grains. Conidia produced using these substrates were tested for virulence against Ae. aegypti larvae obtained from field collected eggs. Larvae (2nd - 3rd instar) were exposed to aqueous conidial suspensions and survival monitored over 7 days. Survival analysis was performed using Log-Rank and Kaplan Meier tests, while fungal growth and conidial yields were analyzed using a two-way ANOVA. RESULTS: There were only small differences between growth rates on RYA and SDA; however, ESALQ 818 showed the highest conidial yield on rice. Conidia produced on rice grains were more virulent, rapidly reducing survival rates of mosquito larvae. ESALQ 818 conidia produced on rice grains, RYA and SDA killed 100% of the larvae on the 2nd, 3rd and 4th day of exposure, respectively. IP 46 virulence of was consistently lower than ESALQ 818 for all the media tested. INTERPRETATION & CONCLUSION: The choice of culture media can influence the virulence of fungal conidia to Ae. aegypti larvae, demonstrating the importance of not only selecting the most virulent isolate but also standardizing growth conditions when screening for virulence.

Morphine administered post-trial induces potent morphine conditioned effects if the context is novel but not if the context is familiar.

Morphine administered shortly after exposure to a novel environment induces potent locomotor stimulant conditioning. Environmental novelty is important as pre-exposure (PE) to a stimulus can attenuate the capacity to acquire conditioned stimulus (CS). Here, the importance of environmental novelty for the efficacy of an open-field to become a CS for elicitation of a morphine conditioned response was assessed by comparing the effects of morphine administered post-trial following a 5 min exposure to a novel environment versus a PE environment. Four groups of rats (2 vehicle and 2 morphine groups) were used. Two groups received ten daily 5 min non-drug PEs to an open-field arena and the other two groups were not pre-exposed to the environment. Subsequently, all groups received post-trial injections of either vehicle or morphine immediately after each of five daily 5 min sessions in the open-field. Importantly, on the first day of testing prior to the first post-test morphine administration, the locomotor activity of the novel and PE groups was not different. Over the 5 post-trial morphine treatments, the activity of the PE morphine group, the PE vehicle and the novel environment vehicle groups did not change and were equivalent. In contrast, in the novel environment morphine group, a conditioned hyper-activity response increased with repeated post-trial morphine treatments. For the morphine group it is suggested that the novel environment initiated a post-trial stimulus trace that occurred in temporal contiguity with the post-trial drug response and enabled the trace to become a CS for the morphine unconditioned response. In contrast, PE induced a latent inhibition effect in the PE morphine group, thus the post-trial CS trace was insufficient to become associated to the morphine response and no conditioning occurred. In addition to conventional drug induced Pavlovian delay conditioning, the findings are suggestive of drug induced Pavlovian trace conditioning.

Post-trial low dose apomorphine prevents the development of morphine sensitization.

The development of sensitization is one of the hallmarks of addictive drugs. Consistent with this relationship many studies have demonstrated that the highly addictive opioid agonist morphine induces sensitization effects. In this study, we administered morphine (10 mg/kg) (MOR) to induce sensitization. In that sensitization is considered to involve associative processes and that dopamine activity is an important contributor to learning and memory processes, we administered a dopamine inhibitory treatment using apomorphine (0.05 mg/kg) (APO) during memory consolidation following a morphine sensitization treatment protocol. Seemingly, a decrease in dopamine activity during consolidation would impair the salience of the association of the morphine response with the contextual cues during consolidation and interfere with the development of morphine sensitization. In two separate experiments, MOR or vehicle (VEH) were administered pre-trial and either VEH or APO were administered post-trial over 5 and 10 days of treatment, respectively. In both the 5 and 10 drug treatment sessions post-trial experiments, MOR groups given VEH immediately post-trial exhibited strong sensitization effects. These sensitization effects were substantially attenuated in the MOR groups given APO immediately post-trial but not in the MOR groups given APO after a 15 min. post-trial delay. In subsequent conditioning and sensitization challenge tests, the MOR groups that had been given APO immediately post-trial exhibited diminished sensitization and conditioned responses relative to MOR groups that had received VEH or APO delayed post-trial. This MOR-APO interaction effect was unique in that it occurred post-trial so that it was only expressed in a pre-trial test in which only MOR was administered. Seemingly, the inhibitory dopamine effect of APO was incorporated into memory during the post-trial consolidation process suggesting that drug/drug interactions can occur during consolidation.

Morphine administered post-trial can induce potent conditioned morphine effects.

Morphine has substantial pro-dopamine effects and in rodents, this is expressed in behavior as increased locomotor activation. Here we administered post-trial 3 dose levels of morphine (3.0, 5.0 and 10.0 mg/kg) or vehicle either immediately or after a 15 min delay to different groups of rats following a brief (5 min) exposure to a novel test environment. Three post-trial injections were administered on three successive days. One day after the first post-trial morphine injections, the non-drug activity levels in the immediate post-trial morphine treatment groups were selectively increased compared to vehicle groups. The activity effects were potentiated with repeated immediate post-trial morphine treatments but the same morphine treatments given after a 15 min post-trial delay did not increase activity in any tests and did not differ from vehicle. Subsequently, all groups were given 5 daily non-drug test sessions as an extinction protocol. The increased activity levels in the 5.0 and 10.0 mg/kg immediate post-trial morphine groups were sustained over the five extinction sessions. Two days later all groups were given a 30 min non-drug test and the 5.0 and 10.0 immediate post-trial groups continued to exhibit a heightened level of activity relative to vehicle restricted to the initial 10 min of the test session. There were no other group differences. The findings that the locomotor stimulant effects in the immediate post-test morphine groups occurred on non-drug tests and that the same morphine treatments given 15 min post-test were without effect are consistent with a conditioned morphine effect. In that acquisition of familiarization with a new environment is a basic learning process that engages consolidation mechanisms, it is possible that the immediate post-trial morphine effects that occur concurrently with consolidation can become incorporated into this consolidation process and subsequently be expressed as a conditioned drug effect.

Medial prefrontal cortex ERK and conditioning: Evidence for the association of increased medial prefrontal cortex ERK with the presence/absence of apomorphine conditioned behavior using a unique post-trial conditioning/extinction protocol.

Increases in medial prefrontal cortex ERK have been linked to learning and memory processes. In the present study separate groups of rats initially underwent testing in an open-field paired with either 2.0 mg/kg apomorphine or vehicle injections. Subsequently, in a brief conditioning 5 min. test the paired apomorphine group manifested a conditioned hyperactivity response. The vehicle/apomorphine groups were then subdivided into two vehicle and two apomorphine subgroups matched for their activity scores in this conditioning test. Following another apomorphine/vehicle pairing in the test environment the groups received 3 additional 5 min. non-drug conditioning tests in which the groups received post-trial vehicle/apomorphine treatments. The vehicle groups received vehicle either immediately or 15 min. after the first two of the three conditioning tests and the apomorphine groups received 2.0 mg/kg either immediately or 15 min. after the first two of the three conditioning tests. In the first conditioning test both of the apomorphine groups exhibited equivalent conditioned responses. By the third test, the conditioned response of the immediate post-trial apomorphine group remained robust whereas conditioned response of the 15 min. apomorphine post-trial group was extinguished. Immediately following the third conditioning test, the animals were euthanized and ERK was measured in the medial prefrontal cortex and the nucleus accumbens. ERK was enhanced in both brain areas, selectively in the immediate apomorphine post-trial group. Increased ERK activity linked to the presence of the apomorphine conditioned response coupled with the absence of increased ERK activity following extinction of the apomorphine conditioned response suggests that ERK activity immediately following a conditioning test is an indicator of activity in brain systems with substantial dopaminergic input that are important in learning and memory. The facilitative effects of the immediate post-trial apomorphine treatment on the conditioned response are also consistent with the proposition that immediate post-trial dopaminergic drug treatments can modify the re-consolidation of conditioned behavior.

Reversal of morphine conditioned behavior by an anti-dopaminergic post-trial drug treatment during re-consolidation.

Morphine has potent pro-dopamine effects that can be manifested as hyper-locomotion and these behavioral effects can undergo conditioning and sensitization. The aim of the present study was to assess whether an inhibitory dopaminergic post-trial treatment (0.05 mg/kg apomorphine) given during re-consolidation could reduce morphine conditioning. To induce conditioned morphine hyperactivity and control for morphine exposure, a paired/unpaired Pavlovian conditioning protocol was used. The morphine paired groups received morphine in the open-field test arena and the unpaired groups received the same morphine (10 mg/kg) treatments but in a different environment. The morphine treatments were administered once per day for 5 days. With repeated treatments, the paired morphine groups developed a sensitized hyper-locomotion response whereas the unpaired morphine groups did not differ from vehicle groups. Subsequently, the paired, unpaired and vehicle groups were given four daily non-drug 5 min conditioning tests. In these conditioning tests, the paired but not the unpaired and vehicle groups exhibited a conditioned locomotor stimulant response. These groups were subdivided into matched groups and received either vehicle or 0.05 mg/kg apomorphine either during re-consolidation immediately post-test or after re-consolidation 15 min post-test. In the immediate post-trial treatment groups, the morphine conditioned response in the paired group was eliminated after only one post-trial apomorphine treatment. The same immediate 0.05 mg/kg apomorphine post-trial treatments had no effect on the unpaired morphine or vehicle groups. In the paired group that received vehicle immediately post-trial, the conditioned response remained robust and unchanged over the four conditioning tests. In the post-trial 15 min delay treatment groups, the post-trial apomorphine treatments had no effect on the morphine conditioned response. These results showed that the inhibition of dopamine activity by apomorphine during the re-consolidation of a cue activated morphine conditioned response eliminated morphine conditioned effects. In that morphine conditioned effects are important for the initiation of addiction and in triggering drug craving and relapse, this finding has potential relevance to opioid addiction treatment.

ERK activation in the prefrontal cortex by acute apomorphine and apomorphine conditioned contextual stimuli.

The activation of extracellular signal-regulated kinase protein (ERK) has been linked to the adaptive responses to environmental changes and memory. The aim of this study was to measure ERK activation in primary dopamine projection areas namely, the prefrontal cortex and the nucleus accumbens, following a conditioned dopaminergic drug response. Initially, the effect of unconditioned apomorphine (2.0mg/kg) administration on ERK activation was measured and the results showed an increase in ERK for both brain regions. Subsequently, two experiments were conducted to assess ERK activation in these two areas following apomorphine conditioned contextual stimuli. In experiment 1, rats received 5 daily injections of 2.0mg/kg apomorphine or vehicle immediately prior to placement in an open-field. After a withdrawal period of two days, a conditioning test was conducted, in which rats received a 30min non-drug test. Immediately after completion of the test, an immunohistochemical protocol was carried out to measure ERK activation. In experiment 2, a similar test protocol was performed except that the treatments were administered 30min following open-field tests (post-trial experiment). The results showed that the repeated apomorphine treatments given prior to testing induced conditioned effects. An increase in ERK activation was seen in the prefrontal cortex but not in the nucleus accumbens. There was no conditioning response observed in the post-trial experiment and no differential ERK activation. These observations implicate the prefrontal cortex in the associative neuro-adaptive changes induced by dopaminergic stimulation.

Larvicidal Potential of the Halogenated Sesquiterpene (+)-Obtusol, Isolated from the Alga Laurencia dendroidea J. Agardh (Ceramiales: Rhodomelaceae), against the Dengue Vector Mosquito Aedes aegypti (Linnaeus) (Diptera: Culicidae).

Dengue is considered a serious public health problem in many tropical regions of the world including Brazil. At the moment, there is no viable alternative to reduce dengue infections other than controlling the insect vector, Aedes aegypti Linnaeus. In the continuing search for new sources of chemicals targeted at vector control, natural products are a promising alternative to synthetic pesticides. In our work, we investigated the toxicity of a bioactive compound extracted from the red alga Laurencia dendroidea J. Agardh. The initial results demonstrated that crude extracts, at a concentration of 5 ppm, caused pronounced mortality of second instar A. aegypti larvae. Two molecules, identified as (-)-elatol and (+)-obtusol were subsequently isolated from crude extract and further evaluated. Assays with (-)-elatol showed moderate larvicidal activity, whereas (+)-obtusol presented higher toxic activity than (-)-elatol, with a LC50 value of 3.5 ppm. Histological analysis of the larvae exposed to (+)-obtusol revealed damage to the intestinal epithelium. Moreover, (+)-obtusol-treated larvae incubated with 2 µM CM-H2DCFDA showed the presence of reactive oxygen species, leading us to suggest that epithelial damage might be related to redox imbalance. These results demonstrate the potential of (+)-obtusol as a larvicide for use against A. aegypti and the possible mode of action of this compound.

Reciprocal activation/inactivation of ERK in the amygdala and frontal cortex is correlated with the degree of novelty of an open-field environment.

RATIONALE: Phosphorylated extracellular signal-regulated kinase (ERK) has been used to identify brain areas activated by exogenous stimuli including psychostimulant drugs. OBJECTIVE: Assess the role of the amygdala in emotional responses. METHODS: Experimental manipulations were performed in which environmental familiarity was the variable. To provide the maximal degree of familiarity, ERK was measured after removal from the home cage and re-placement back into the same cage. To maximize exposure to an unfamiliar environment, ERK was measured following placement into a novel open field. To assess whether familiarity was the critical variable in the ERK response to the novel open field, ERK was also measured after either four or eight placements into the same environment. ERK quantification was carried out in the amygdala, frontal cortex, and the nucleus accumbens. RESULTS: After home cage re-placement, ERK activation was found in the frontal cortex and nucleus accumbens but was absent in the amygdala. Following placement in a novel environment, ERK activation was more prominent in the amygdala than the frontal cortex or nucleus accumbens. In contrast, with habituation to the novel environment, ERK phosphors declined markedly in the amygdala but increased in the frontal cortex and nucleus accumbens to the level observed following home cage re-placement. CONCLUSIONS: The differential responsiveness of the amygdala versus the frontal cortex and the nucleus accumbens to a novel versus a habituated environment is consistent with a reciprocal interaction between these neural systems and points to their important role in the mediation of behavioral activation to novelty and behavioral inactivation with habituation.

Increase in medial frontal cortex ERK activation following the induction of apomorphine sensitization.

Repeated high dose injections of the direct acting D1/D2 agonist apomorphine (APO) induces context specific behavioral sensitization. We assessed the effects of 2.0 mg/kg APO on open-field locomotor responses of rats over a 30 min period following either single or five daily APO injections. Acute injections increased locomotor activity, which was markedly increased in rats given 5 daily APO injections. This progressive increase in locomotion during the repeated APO treatments is indicative of behavioral sensitization. Immediately following the open-field test for the acute and the fifth apomorphine injection, the animals were euthanized and their brain tissue was prepared for immunohistochemistry. ERK immunoreactive nuclei in the medial prefrontal cortex (PFC), nucleus accumbens (NAcc), amygdala (AMYG) and lateral hypothalamus (LH) were quantified. The acute apomorphine injections increased ERK in all brain areas as compared to vehicle. Following the fifth apomorphine injection, ERK significantly increased in the PFC, decreased in the amygdala but was unchanged in the LH and NAcc. The selective increase in ERK activity in the PFC associated with behavioral sensitization, points to a possible pivotal role of the dopamine projection to the medial frontal cortex in the mediation of neural plasticity, considered to underlie the sensitization processes induced by dopaminergic drugs.