Pre-exposed Animals Research Articles

Considerations for bioaccumulation studies in fish with nanomaterials

Nanomaterials (NMs) pose challenges in performing bioaccumulation studies in fish and in regulatory interpretation of results. Therefore, a clear guidance is needed to obtain reliable, reproducible and comparable results. By analysing all the available literature, we aim in this manuscript to identify the critical aspects that should be addressed in these type of studies. Seventy-eight studies from a total of 67 published articles were identified in which a variety of approaches were used: aqueous exposure (49 studies), dietary exposure (19), and pre-exposed animals for trophic transfer studies (10). The NMs tested included TiO2, Zn, ZnO, Cu, CuO, Ag, Au, CeO2, Fe2O3, Fe3O4, Se, CdS, CdSe/ZnS-QDs, CdTe/ZnS-QDs, graphene, fullerenol and MWCNTs. In general, there is a scarcity of bioaccumulation studies for the different NMs. In particular, studies that use the dietary exposure route are lacking. TiO2 NMs are the most studied for bioaccumulation potential in fish (20%), whereas very few data were available for CuO, FeO and carbon-based NMs. Different information gaps were identified in these studies that hamper overall conclusions to be made on the bioaccumulation potential of NMs. The main critical issues related to NM testing for bioaccumulation include: maintenance of stable exposure concentrations, the influence of feeding regimen on uptake and elimination, the use of appropriate feed spiking methodologies, the potential need for testing different concentrations, and the reporting of bioaccumulation endpoints (BCF/BMF). Each of these issues needs further guidance to allow proper use and reporting of NM bioaccumulation data for regulatory purposes.

Immunoinformatics Evaluation of a Fusion Protein Composed of Leishmania infantum LiHyV and Phlebotomus kandelakii Apyrase as a Vaccine Candidate against Visceral Leishmaniasis

Background:Visceral leishmaniasis (VL) is a lethal parasitic disease, transmitted by sand fly vectors. Immunomodulatory properties of sand fly saliva proteins and their protective effects against Leishmania infection in pre-exposed animals suggest that a combination of an antigenic salivary protein along with a Leishmania antigen can be considered for designing a vaccine against leishmaniasisMethods:Three different fusion forms of L. infantum hypothetical protein (LiHyV) in combination with Phlebotomus kandelakii salivary apyrase (PkanAp) were subjected to insilico analyses. Major Histocompatibility Complex (MHC) class I and II epitopes in both humans and BALB/c mice were predicted. Antigenicity, immunogenicity, epitope conservancy, toxicity, and population coverage were also evaluated.Results:Highly antigenic promiscuous epitopes consisting of truncated LiHyV (10–285) and full-length PkanAp (21–329) were identified in human and was named Model 1. This model contained 25 MHC-I and 141 MHC-II antigenic peptides which among them, MPANSDIRI and AQSLFDFSGLALDSN were fully conserved. LALDSNATV, RCSSALVSI, ALVSINVPL, SAVESGALF of MHC-I epitopes, and 28 MHC-II binding epitopes showed 60% conservancy among various clades. A population coverage with a rate of >75% in the Iranian population and >70% in the whole world was also identified.Conclusion:Based on this in-silico approach, the predicted Model 1 could potentially be used as a vaccine candidate against VL.

Exposure-related, global alterations in innate and adaptive immunity; a consideration for re-use of non-human primates in research.

BackgroundNon-human primates (NHPs) play an important role in biomedical research, where they are often being re-used in multiple research studies over the course of their life-time. Researchers employ various study-specific screening criteria to reduce potential variables associated with subsequent re-use of NHPs. However, criteria set for NHP re-assignments largely neglect the impact of previous exposures on overall biology. Since the immune system is a key determinant of overall biological outcome, an altered biological state could be predicted by monitoring global changes in the immune profile. We postulate that every different exposure or a condition can generate a unique global immune profile in NHPs.MethodsChanges in the global immune profile were evaluated in three different groups of rhesus macaques previously enrolled in dengue or malaria vaccine studies over six months after their last exposure. Naïve animals served as the baseline. Fresh blood samples were stained with various immune cell surface markers and analyzed by multi-color flow-cytometry to study immune cell dynamics in the peripheral blood. Serum cytokine profile in the pre-exposed animals were analyzed by mesoscale assay using a customized U-PLEX NHP biomarker panel of 12 cytokines/chemokines.ResultsPre-exposed macaques showed altered dynamics in circulating cytokines and certain innate and adaptive immune cell subsets such as monocytes, HLA-DR+NKT cells, B cells and T cells. Some of these changes were transient, while some lasted for more than six months. Each group seemed to develop a global immune profile unique to their particular exposure.ConclusionOur data strongly suggest that re-used NHPs should be evaluated for long-term, overall immunological changes and randomly assigned to new studies to avoid study bias.

Establishment, optimisation and quantitation of a bioluminescent murine infection model of visceral leishmaniasis for systematic vaccine screening

Visceral leishmaniasis is an infectious parasitic disease caused by the protozoan parasites Leishmania donovani and Leishmania infantum. The drugs currently used to treat visceral leishmaniasis suffer from toxicity and the emergence of parasite resistance, and so a better solution would be the development of an effective subunit vaccine; however, no approved vaccine currently exists. The comparative testing of a large number of vaccine candidates requires a quantitative and reproducible experimental murine infection model, but the parameters that influence infection pathology have not been systematically determined. To address this, we have established an infection model using a transgenic luciferase-expressing L. donovani parasite and longitudinally quantified the infections using in vivo bioluminescent imaging within individual mice. We examined the effects of varying the infection route, the site of adjuvant formulation administration, and standardised the parasite preparation and dose. We observed that the increase in parasite load within the liver during the first few weeks of infection was directly proportional to the parasite number in the initial inoculum. Finally, we show that immunity can be induced in pre-exposed animals that have resolved an initial infection. This murine infection model provides a platform for systematic subunit vaccine testing against visceral leishmaniasis.

Test-Retest Repeatability of [18F]MC225-PET in Rodents: A Tracer for Imaging of P-gp Function.

In longitudinal PET studies, animals are repeatedly anesthetized which may affect the repeatability of PET measurements. The aim of this study was to assess the effect of anesthesia on the P-gp function as well as the reproducibility of [18F]MC225 PET scans. Thus, dynamic PET scans with blood sampling were conducted in 13 Wistar rats. Seven animals were exposed to isoflurane anesthesia 1 week before the PET scan (“Anesthesia-exposed” PET). A second group of six animals was used to evaluate the reproducibility of measurements of P-gp function at the blood–brain barrier (BBB) with [18F]MC225. In this group, two PET scans were made with a 1 week interval (“Test” and “Retest” PET). Pharmacokinetic parameters were calculated using compartmental models and metabolite-corrected plasma as an input function. “Anesthesia-exposed” animals showed a 28% decrease in whole-brain volume of distribution (VT) (p < 0.001) compared to “Test”, where the animals were not previously anesthetized. The VT at “Retest” also decreased (19%) compared to “Test” (p < 0.001). The k2 values in whole-brain were significantly increased by 18% in “Anesthesia-exposed” (p = 0.005) and by 15% in “Retest” (p = 0.008) compared to “Test”. However, no significant differences were found in the influx rate constant K1, which is considered as the best parameter to measure the P-gp function. Moreover, Western Blot analysis did not find significant differences in the P-gp expression of animals not pre-exposed to anesthesia (“Test”) or pre-exposed animals (“Retest”). To conclude, anesthesia may affect the brain distribution of [18F]MC225 but it does not affect the P-gp expression or function.

Ethanol pre-exposure does not increase delay discounting in P rats, but does impair the ability to dynamically adapt behavioral allocation to changing reinforcer contingencies

Increased subjective discounting of delayed rewards is associated with substance abuse, and individuals tend to discount their drug of choice at a greater rate compared to monetary rewards. While there is evidence indicating that increased delay discounting (DD) is a risk factor for substance abuse, some results suggest that exposure to drugs of abuse also increases DD, but effects are mixed. The current study examined whether ethanol pre-exposure increases DD and if an ethanol reinforcer would be discounted at a greater rate than sucrose. Alcohol preferring (P) rats were pre-exposed to either ethanol or sucrose using an intermittent access protocol (IAP) for 8 weeks. Then animals completed an operant fixed choice procedure where each pre-exposure group was split into either an ethanol or sucrose reinforcer group. Afterwards, animals completed an adjusting delay DD task using the same groups as the fixed choice task. Animals that received access to ethanol in the IAP showed increased delayed reward preference in a delay and session dependent manner. Specifically, ethanol pre-exposed animals took more sessions to decrease their preference for the delayed reward at longer delays. In the adjusting delay task, no differences in mean adjusting delays were seen, but ethanol pre-exposure impaired animals' ability to reach stability criteria. The observed results are not consistent with ethanol pre-exposure causing a change in DD. Rather they indicate ethanol pre-exposure impaired animals' ability to reallocate their behavior in response to a change in reinforcer contingencies. The current findings extend prior results showing alcohol naïve P rats exhibit both increased DD and decreased response inhibition (Beckwith and Czachowski 2014, 2016) by demonstrating that after alcohol exposure they exhibit a form of behavioral inflexibility. Hence, a “two-hit” genetic vulnerability/environmental acceleration of addictive behavior is supported.

Multiple tracking and machine learning reveal dopamine modulation for area-restricted foraging behaviors via velocity change in Caenorhabditis elegans

Food exploration is an essential survival behavior in organisms. To find food efficiently, many organisms use a foraging strategy called area-restricted search (ARS) wherein individuals first turn more frequently, restricting their search to one area, then turn less frequently, moving along a straight path to widen the search area. Previous research suggests that the nematode Caenorhabditis elegans shows ARS behavior by changing turn frequency, and that dopamine is a crucial determinant. However, the effects of dopamine on multiple behavioral parameters have remained unknown. Here, we evaluated turn (pirouette) frequency, moving velocity, and specific area occupancy (cell occupancy) over time by using a multiple-worms tracking system. In the control (mock) experiments, all parameters changed over time, but no changes were observed in experiments with dopamine pre-exposed and dopamine-deficient animals. In inverse reinforcement learning analysis, the value function for specific velocity was found to modulate over time in mock animals only. These results demonstrate that dopamine regulates ARS via changes not only to pirouette frequency change but also to velocity.

Shedding light on the effects of hydraulic fracturing flowback and produced water on phototactic behavior in Daphnia magna

The effluent produced during hydraulic fracturing (i.e. flowback and produced water; FPW), is a complex hyper-saline solution that is known to negatively impact the survival and the fitness of the water flea Daphnia magna, but to date effects on behavior are unstudied. In the current study, the effects of FPW on phototactic behavior of D. magna were examined. Exposure of naïve animals to FPW resulted in a dose-dependent increase in the speed of appearance of daphnids in the illuminated zone of the test apparatus (i.e. a faster positive phototaxis response). A similar dose-dependent response was observed in a test solution where the salt content of FPW was recreated in the absence of other components, suggesting that the effect was largely driven by salinity. The effect of FPW was significant when the raw FPW sample was diluted to 20% of its initial strength, while the effect of salt-matched solution was significant at a 10% dilution. A distinct effect was observed following FPW pre-exposure. After a 24 h pre-exposure to 1.5% FPW, Daphnia displayed a significantly inhibited positive phototaxis response when examined in control water, relative to control animals that were not pre-exposed to FPW. This effect was not observed in salinity pre-exposed animals, however these daphnids displayed a significantly reduced phototactic response when tested in saline waters, indicating a loss of the positive phototaxis seen in naïve organisms. These data indicate that FPW can induce perturbations in the behavior of aquatic invertebrates, an effect that may influence processes such as feeding and predation rates.

Oxidative stress and neurobehavioural changes in rats following copper exposure and their response to MiADMSA andd-penicillamine

An increase in copper concentration in body may lead to hepatolenticular degeneration which is considered as one clinical feature of Wilson’s disease. Chelation therapy using d-penicillamine is the preferred medical treatment for reducing the toxic effects of copper. However, a few shortcomings associated with d-penicillamine led us to search of an alternative antidote for copper toxicity. Monoisoamyl-2, 3-dimercaptosuccinic acid (MiADMSA), a potent arsenic chelator under clinical trial, has been reported to reduce system copper level. Thus, the present study was envisaged to explore the ameliorative effect of MiADMSA against copper toxicity. Copper pre-exposed animals (CuSO4.5H2O; 100 mg/kg; p.o., for 6 weeks) were segregated in different groups and were administered equimolar dose (0.3 mEq/kg/day; p.o.) of d-penicillamine and MiADMSA for 5 days. The effect of different treatments on spontaneous locomotor activity, muscle coordination, depression like behaviour and contextual fear memory was analysed using neurobehavioural battery test. Biochemical variables related to oxidative stress, zinc and copper concentration were determined in liver, kidney and brain. The results suggested that copper exposure led to oxidative stress in liver, kidney and blood, along with moderate effects in brain. Treatment with d-penicillamine and MiADMSA reduced liver copper load. MiADMSA produced more pronounced beneficial effect compared to d-penicillamine by increasing brain GPx activity. Our study suggests that MiADMSA might be equally effective as d-penicillamine in depleting body copper load. More detailed studies using different doses are required to suggest whether MiADMSA could be an alternative for d-penicillamine in reducing oxidative injury, neurobehavioural changes and depleting body copper burden.

Behavioral predictors of alcohol drinking in a neurodevelopmental rat model of schizophrenia and co-occurring alcohol use disorder

Alcohol use disorder commonly occurs in patients with schizophrenia and contributes greatly to its morbidity. Unfortunately, the neural and behavioral underpinnings of alcohol drinking in these patients are not well understood. In order to begin to understand the cognitive and reward-related changes that may contribute to alcohol drinking, this study was designed to address: 1) latent inhibition; 2) conditioning; and 3) extinction of autoshaping in a neurodevelopmental rat model with relevance to co-occurring schizophrenia and alcohol use disorders, the neonatal ventral hippocampal lesioned (NVHL) rat. NVHL lesions (or sham surgeries) were performed on post-natal day 7 (PND7) and animals were given brief exposure to alcohol during adolescent (PND 28–42). Latent inhibition of autoshaping, conditioning and extinction were assessed between PND 72–90. On PND90 animals were given alcohol again and allowed to establish stable drinking. Latent inhibition of autoshaping was found to be prolonged in the NVHL rats; the NVHL rats pre-exposed to the lever stimulus were slower to acquire autoshaping than sham pre-exposed rats. NVHL rats that were not pre-exposed to the lever stimulus did not differ during conditioning, but were slower to extinguish conditioned responding compared to sham controls. Finally, the NVHL rats from both groups drank significantly more alcohol than sham rats, and the extent of latent inhibition predicted future alcohol intake in the pre-exposed animals. These findings suggest that the latent inhibition of autoshaping procedure can be used to model cognitive- and reward-related dysfunctions in schizophrenia, and these dysfunctions may contribute to the development of co-occurring alcohol use.

Repeated ventral midbrain neurotensin injections sensitize to amphetamine-induced locomotion and ERK activation: A role for NMDA receptors

Previous studies have shown that activation of ventral midbrain NMDA receptors is required to initiate sensitization by amphetamine. In view of the recent evidence that neurotensin modulates ventral midbrain glutamate neurotransmission, we tested the hypothesis that neurotensin is acting upstream to glutamate to initiate sensitization to the behavioral and neurochemical effects of amphetamine. During a first testing phase, adult male rats implanted with bilateral ventral midbrain cannulae were injected every second day for three days with D-[Tyr11]neurotensin (1.5 nmol/side), the preferred NMDA GluN2A/B antagonist, CPP (40 or 120 pmol/side), the selective GluN2B antagonist, Ro04-5595 (200 or 1200 pmol/side), CPP (40 or 120 pmol/side) + D-[Tyr11]neurotensin (1.5 nmol/side) or Ro04-5595 (200 or 1200 pmol/side) + D-[Tyr11]neurotensin (1.5 nmol/side) and locomotor activity was measured immediately after the injection. Five days after the last central injection, the locomotor response or the expression of phosphorylated extracellular signal-regulated kinases 1/2 (pERK1/2) in neurons of different limbic nuclei was measured following a systemic injection of amphetamine sulfate (0.75 mg/kg, i.p.). Results show that amphetamine induced significantly stronger locomotor activity and pERK1/2 expression in the nucleus accumbens shell and infralimbic cortex in neurotensin pre-exposed animals than in controls (vehicle pre-exposed). These sensitization effects initiated by neurotensin were prevented by CPP, but not Ro04-5595. These results support the hypothesis that neurotensin is stimulating glutamate neurotransmission to initiate neural changes that sub-serve amphetamine sensitization and that glutamate is acting on NMDA receptors that are mostly likely composed of GluN2A, but not GluN2B, subunits.This article is part of the Special Issue entitled ‘Ionotropic glutamate receptors’.

Ciguatoxin reduces regenerative capacity of axotomized peripheral neurons and delays functional recovery in pre-exposed mice after peripheral nerve injury.

Ciguatera fish poisoning (CFP) results from consumption of tropical reef fish containing ciguatoxins (CTXs). Pacific (P)-CTX-1 is among the most potent known CTXs and the predominant source of CFP in the endemic region responsible for the majority of neurological symptoms in patients. Chronic and persistent neurological symptoms occur in some CFP patients, which often result in incomplete functional recovery for years. However, the direct effects of exposure to CTXs remain largely unknown. In present study, we exposed mice to CTX purified from ciguatera fish sourced from the Pacific region. P-CTX-1 was detected in peripheral nerves within hours and persisted for two months after exposure. P-CTX-1 inhibited axonal regrowth from axotomized peripheral neurons in culture. P-CTX-1 exposure reduced motor function in mice within the first two weeks of exposure before returning to baseline levels. These pre-exposed animals exhibited delayed sensory and motor functional recovery, and irreversible motor deficits after peripheral nerve injury in which formation of functional synapses was impaired. These findings are consistent with reduced muscle function, as assessed by electromyography recordings. Our study provides strong evidence that the persistence of P-CTX-1 in peripheral nerves reduces the intrinsic growth capacity of peripheral neurons, resulting in delayed functional recovery after injury.

Latent inhibition of conditioned taste aversion in rats with amygdaloid or hippocampal lesions

The amygdala and hippocampus play crucial roles in the latent inhibition of different conditioned responses, such as fear conditioned. Nevertheless, the involvement of these structures in the latent inhibition of conditioned taste aversion (CTA) is uncertain. In the present study, we explore the relevance of the amygdala and hippocampus in latent inhibition using a CTA paradigm. The effects of taste pre-exposures vs. non-pre-exposures on taste aversion conditioning were compared in Wistar rats with amygdaloid or hippocampal excitotoxic lesions. All pre-exposed animals consumed significantly more on the test day than did non-pre-exposed animals. Therefore, neither amygdaloid nor hippocampal lesions prevented the latent inhibition phenomenon. However, the expected taste neophobia was potentially affected by lesions to either the amygdala or hippocampus. These findings suggest that the amygdala and hippocampus seem to be necessary for taste neophobia but not for the acquisition of latent inhibition of CTA. The differential involvement of both structures in latent inhibition under different associative learning paradigms is briefly discussed.

Habituation of the responsiveness of mesolimbic and mesocortical dopamine transmission to taste stimuli

The presentation of novel, remarkable, and unpredictable tastes increases dopamine (DA) transmission in different DA terminal areas such as the nucleus accumbens (NAc) shell and core and the medial prefrontal cortex (mPFC), as estimated by in vivo microdialysis studies in rats. This effect undergoes adaptive regulation, as there is a decrease in DA responsiveness after a single pre-exposure to the same taste. This phenomenon termed habituation has been described as peculiar to NAc shell but not to NAc core and mPFC DA transmission. On this basis, it has been proposed that mPFC DA codes for generic motivational stimulus value and, together with the NAc core DA, is more consistent with a role in the expression of motivation. Conversely, NAc shell DA is specifically activated by unfamiliar or novel taste stimuli and rewards, and might serve to associate the sensory properties of the rewarding stimulus with its biological effect (Bassareo etal., 2002; Di Chiara etal., 2004). Notably, habituation of the DA response to intraoral sweet or bitter tastes is not associated with a reduction in hedonic or aversive taste reactions, thus indicating that habituation is unrelated to satiety-induced hedonic devaluation and that it is not influenced by DA alteration or depletion. This mini-review describes specific circumstances of disruption of the habituation of NAc shell DA responsiveness (De Luca etal., 2011; Bimpisidis etal., 2013). In particular, we observed an abolishment of NAc shell DA habituation to chocolate (sweet taste) by morphine sensitization and mPFC 6-hydroxy-dopamine hydrochloride (6-OHDA) lesion. Moreover, morphine sensitization was associated with the appearance of the habituation in the mPFC, and with an increased and delayed response of NAc core DA to taste in naive rats, but not in pre-exposed animals. The results here described shed light on the mechanism of the habituation phenomenon of mesolimbic and mesocortical DA transmission, and its putative role as a marker of cortical dysfunction in specific conditions such as addiction.

Mechanical Compression and Nucleus Pulposus Application on Dorsal Root Ganglia Differentially Modify Evoked Neuronal Activity in the Thalamus

A combination of mechanical compression caused by a protruding disc and leakage of nucleus pulposus (NP) from the disc core is presumed to contribute to intervertebral disc hernia-related pain. Experimental models of disc hernia including both components have resulted in changes in neuronal activity at the level of the dorsal root ganglion (DRG) and spinal cord, but changes within the brain have been less well studied. However, acute application of NP to a DRG without mechanical compression rapidly increases neuronal activity in the thalamus, a major brain relay nucleus processing information from sensory pathways including ascending nociceptive tracts. The combination of mechanical compression and NP might therefore result in further increases in central neuronal activity. Using an experimental disc herniation rat model including both mechanical compression and NP the present study aimed to investigate changes in neuronal activity in the contralateral thalamic ventral posterior lateral nucleus in vivo. Measurements were obtained while electrically stimulating the ipsilateral sciatic nerve at Aδ fiber intensities. The L4 DRG was subjected to light mechanical compression and NP exposure, and acute changes in evoked thalamic responses were recorded for up to 40 min. In order to compare effects in naïve animals with effects following a longer period of NP exposure, animals that were either disc-punctured or sham-operated 24 h previously were also included. In all animals, light mechanical compression of the DRG depressed the number of evoked neuronal responses. Prior NP exposure resulted in less potent changes following mechanical compression (80% of baseline) than that observed in naïve animals (50%). During the subsequent NP application, the number of evoked responses compared to baseline increased in pre-exposed animals (to 87%) as well as in naïve animals (72%) in which the removal of the mechanical compression resulted in a further increase (106%). The contribution of acute DRG compression and disc material leakage to changes in transmission in central neuronal networks is likely to be complex and to involve both short-term and long-term effects. Since a light mechanical compression may reduce transmission in nociceptive pathways, it is possible that the presence or absence of NP is crucial for pain development in the acute phase of disc herniation.

Cypermethrin Alters the Expression Profile of mRNAs in the Adult Rat Striatum: A Putative Mechanism of Postnatal Pre-exposure Followed by Adulthood Re-exposure-Enhanced Neurodegeneration

This study was undertaken to investigate the effect of cypermethrin on the expression patterns of mRNAs in the striatum of adulthood alone and postnatal pre-exposed followed by adulthood re-exposed rats using discover chips rat microarrays. The expression patterns of V-akt murine thymoma viral oncogene homolog 1, B-cell lymphoma 2 (BCL-2), BCL-2-associated X protein, caspase 1, caspase 9, death-associated protein 3 and interleukin-1β were validated by the qRT-PCR. The expressions of inducible nitric oxide synthase (iNOS) and major histocompatibility complex (MHC) II were assessed immunohistochemically; however, tumour protein p53 and cytochrome c (mitochondrial and cytosolic) expressions were checked at protein level by western blotting. Cypermethrin differentially regulated 65 transcripts at one or the other stage of exposure and 21 transcripts exhibited more pronounced alterations in the postnatal pre-exposed and adulthood re-challenged rats. The results of qRT-PCR were in accordance with the microarray observations and the expressions of iNOS, p53 and cytosolic cytochrome c and MHC II positivity were increased while the level of mitochondrial cytochrome c was reduced in adulthood treated animals. The effects were more pronounced in the postnatal pre-exposed followed by adulthood re-exposed rats. The results obtained thus suggest that multiple pathways are involved in the neurodegeneration as well as in enhancing the vulnerability of neurons in cypermethrin pre-exposed postnatal animals upon re-exposure during adulthood.

Perinatal undernutrition facilitates morphine sensitization and cross-sensitization to cocaine in adult rats: a behavioral and neurochemical study

The development of sensitization to the locomotor effects of morphine and cross-sensitization between morphine and cocaine were evaluated in adult rats submitted to a protein malnutrition schedule from the 14th day of gestation up to 30 days of age (D-rats), and compared with well-nourished animals (C-rats). Dose-response curves to morphine-induced locomotor activity (5, 7.5, 10 or 15 mg/kg, i.p., every other day for 5 days) revealed a shift to the left in D-rats compared to C-rats. This implies that D-rats showed behavioral sensitization to the lower dose of morphine used (5 mg/kg), which was ineffective in C-rats. Furthermore, when a cocaine challenge (10 mg/kg, i.p) was given 48 h after the last morphine administration, only D-rats exhibited cross-sensitization in morphine-pretreated animals (7.5 and 10 mg/kg). In order to correlate the differential response observed with the functioning of the mesocorticolimbic dopaminergic system, extracellular dopamine (DA) levels were measured in the nucleus accumbens (core and shell) and the dorsal caudate-putamen. A challenge with cocaine in morphine pre-exposed animals produced an increase in DA release, but only in the nucleus accumbens “core” of D-rats. Similar DA levels were found in the nucleus accumbens “shell” and in the dorsal caudate-putamen of both groups. Finally, these results demonstrate that D-rats had a lower threshold for developing both a progressive behavioral sensitization to morphine and a cross-sensitization to cocaine. In accordance with these behavioral findings, a higher responsiveness of the nucleus accumbens core, expressed by increased DA levels, both basal and after cocaine challenge, was observed in D-rats.

Novelty-induced arousal enhances memory for cued classical fear conditioning: Interactions between peripheral adrenergic and brainstem glutamatergic systems

Exposure to novel contexts produce heightened states of arousal and biochemical changes in the brain to consolidate memory. However, processes permitting simple exposure to unfamiliar contexts to elevate sympathetic output and to improve memory are poorly understood. This shortcoming was addressed by examining how novelty-induced changes in peripheral and/or central arousal modulates memory for Pavlovian fear conditioning. Male rats were either exposed to the conditioning chamber for 5-min or given no exposure 24 h before conditioning with five tone-shock (0.35 mA) pairings. Retention was assessed 48 h later in a different context. Non-pre-exposed animals exhibited significantly greater freezing during conditioned stimulus (CS) presentations than did pre-exposed animals (P < 0.05). The improvement in retention produced by novelty was attenuated by pretraining a blockade of peripheral beta-adrenergic receptors with sotalol (6 mg/kg, i.p.). Study 2 revealed that novelty-induced increases in peripheral autonomic output are conveyed to the brain by visceral afferents that synapse upon brainstem neurons in the nucleus tractus solitarius (NTS). Blocking AMPA receptor activity in the NTS with CNQX (1.0 microg) significantly reduced freezing to the CS in non-pre-exposed animals (P < 0.01). Study 3 showed that elevating epinephrine levels in habituated animals influences learning through mechanisms similar to those produced by novelty-induced arousal. Pre-exposed animals given epinephrine (0.1 mg/kg) froze significantly more than saline controls (P < 0.01), and this effect was attenuated by intra-NTS infusion of CNQX. The findings demonstrate that novelty-induced arousal or increasing sympathetic activity with epinephrine in pre-exposed animals enhances memory through adrenergic mechanisms initiated in the periphery and transmitted centrally via the vagus/NTS complex.

The Effects of Continuous and Intermittent Ethanol Exposure in Adolesence on the Aversive Properties of Ethanol During Adulthood

Alcohol abuse among adolescents is prevalent. Epidemiological studies suggest that alcohol abuse during the adolescent developmental period may result in long-term changes such as an increased susceptibility to alcohol-related problems in adulthood. Laboratory findings suggest that alcohol exposure during the adolescent developmental period, as compared with adulthood, may differentially impact subsequent neurobehavioral responses to alcohol. The present study was designed to examine whether ethanol exposure, continuous versus intermittent, during the adolescent developmental period would alter the aversive properties of ethanol in adult C3H mice. Periadolescent (PD28) male C3H mice were exposed to 64 hours of continuous or intermittent ethanol vapor. As a comparison, adult (PD70) C3H mice were also exposed to 64 hours of continuous or intermittent ethanol vapor. Six weeks after ethanol exposure, taste aversion conditioning was carried out on both ethanol pre-exposed and ethanol-naive animals using a 1-trial, 1-flavor taste-conditioning procedure. Ethanol exposure during the periadolescent period significantly attenuated a subsequent ethanol-induced conditioned taste aversion, as compared with control animals. Adult animals exposed to chronic ethanol vapor during adolescence showed less of an aversion to an ethanol-paired flavor than ethanol-naive adults. Intermittent exposure to ethanol vapor during periadolescence produced a greater attenuation. It is suggested that ethanol exposure during the periadolescent period results in long-term neurobehavioral changes, which lessen a conditioned aversion to ethanol in adulthood. It is suggested that this age-related effect may underlie the increased susceptibility to alcohol-related problems which is negatively correlated with the age of onset for alcohol abuse.

Short-term pre-exposure increases earthworm tolerance to mercury

To investigate the effects of pre-exposure to mercury, earthworms of the species Eisenia fetida Savigny were exposed to either a non-lethal mercury concentration (22 mg/kg soil), test soil, or cultivation medium for one week prior to toxicity testing with mercury. In subsequent tests with exposure to various concentrations of mercury for 4 weeks the effects on survival, reproduction (cocoon production) and glutathione (GSH) levels were recorded. Pre-exposure to mercury strongly increased survival. The 50% lethal concentrations (LC 50) for pre-exposed and non pre-exposed earthworms were 545 and 170 mg Hg/kg soil, respectively. Glutathione, which is involved in detoxification of metals such as mercury, was analyzed by a standard spectrophotometric method. Earthworms exposed to mercury concentrations above 22 mg/kg experienced a threefold increase in glutathione levels from a background level of approximately 0.62 μmol/g earthworm wet weight. Glutathione levels were similar in pre-exposed and non pre-exposed earthworms, suggesting that increased glutathione levels did not cause the increased survival of pre-exposed animals. Internal concentrations of mercury were measured with an automated atomic absorbance spectrophotometer, and the uptake rate indicated a one-site binding hyperbola in both pre-exposed and non pre-exposed earthworms. Internal concentrations of mercury reached a maximum of about 150 μg Hg/g earthworm wet weight corresponding to soil concentrations of about 740 mg/kg. The shape of the accumulation curves strongly resembled the ones plotted for internal glutathione levels, suggesting that glutathione levels in earthworms are closely related to internal concentrations of mercury. Critical internal concentrations for effects of mercury on earthworms are also discussed.