James S. Miller

Integration of DNA barcoding into an ongoing inventory of complex tropical biodiversity

Inventory of the caterpillars, their food plants and parasitoids began in 1978 for todays Area de Conservacion Guanacaste (ACG), in northwestern Costa Rica. This complex mosaic of 120 000 ha of conserved and regenerating dry, cloud and rain forest over 0–2000 m elevation contains at least 10 000 species of non‐leaf‐mining caterpillars used by more than 5000 species of parasitoids. Several hundred thousand specimens of ACG‐reared adult Lepidoptera and parasitoids have been intensively and extensively studied morphologically by many taxonomists, including most of the co‐authors. DNA barcoding — the use of a standardized short mitochondrial DNA sequence to identify specimens and flush out undisclosed species — was added to the taxonomic identification process in 2003. Barcoding has been found to be extremely accurate during the identification of about 100 000 specimens of about 3500 morphologically defined species of adult moths, butterflies, tachinid flies, and parasitoid wasps. Less than 1% of the species have such similar barcodes that a molecularly based taxonomic identification is impossible. No specimen with a full barcode was misidentified when its barcode was compared with the barcode library. Also as expected from early trials, barcoding a series from all morphologically defined species, and correlating the morphological, ecological and barcode traits, has revealed many hundreds of overlooked presumptive species. Many but not all of these cryptic species can now be distinguished by subtle morphological and/or ecological traits previously ascribed to ‘variation’ or thought to be insignificant for species‐level recognition. Adding DNA barcoding to the inventory has substantially improved the quality and depth of the inventory, and greatly multiplied the number of situations requiring further taxonomic work for resolution.

Prenatal cocaine exposure induces deficits in Pavlovian conditioning and sensory preconditioning among infant rat pups.

Offspring derived from Sprague-Dawley dams that received daily subcutaneous injection of 40 mg/kg.3 cc-1 cocaine hydrochloride (C40) or saline (LC) from Gestational Days 8-20 were tested for first-order Pavlovian conditioning and sensory preconditioning at Postnatal Days 8 (P8), P12, and P21. Although C40 dams gained significantly less weight than LC dams, pup body weights did not differ between the two groups. Significant sensory preconditioning was obtained at P8 and P12 (but not at P21) in LC offspring, confirming previous reports of decline in performance in this task during ontogeny. In contrast, C40 offspring failed to exhibit sensory preconditioning at any test age. In addition, C40 pups tested at P8 did not display significant first-order conditioning. Taken together these results suggest a more general deficit in cognitive functioning rather than a delay in cognitive development in prenatally cocaine-exposed offspring.

Conditioned place preference with morphine: the effect of extinction training on the reinforcing CR.

Rats were injected with either morphine (5 mg/kg) or saline in association with one set of distinct environmental stimuli, and injected with saline in association with a different set of stimuli. After four conditioning trials, animals were given a 15-minute free-choice test to determine which stimulus environment was preferred. Animals displayed CPP as a significant increase in duration spent within the morphine-associated environment, but did not display any change in number of entries into that environment. In contrast, when extinction training was given following CPP, animals displayed a significant decrease in duration spent per entry into the morphine-associated environment, but did not display any change in total duration spent in that environment. These results suggest that assessment of the reinforcing conditioned response (CR) in the CPP model may require measurement of both duration spent in and number of entries into the drug-associated environment.

Repeated testing attenuates conditioned place preference with cocaine.

Cocaine-treated rats acquired a preference for cocaine-associated contextual stimuli (CS) relative to saline-injected control rats. However, when animals were given repeated tests for conditioned place preference intermittent between conditioning trials, they displayed an attenuation in strength of conditioning. This attenuation was not due to pharmacologic tolerance (Experiment 1), but rather reflected a disruption in learning due to exposure to the CS alone (Experiment 2). Like other examples of classical conditioning, the strength of the conditioned response (CR) as assessed by the conditioned place preference model may be influenced by partial reinforcement.

Prenatal exposure to cocaine disrupts cocaine-induced conditioned place preference in rats

Cocaine-induced conditioned place preference (CPP) was tested in adult offspring of Sprague-Dawley dams that had been injected subcutaneously with 40 mg/kg/3cc cocaine HCl (C40) daily from gestational days 8-20, pair-fed (PF) dams injected with saline, and nontreated control (LC) dams. C40 and PF dams gained significantly less weight than LC dams, although offspring body weights did not differ among the three prenatal treatment groups at birth or in adulthood. Significant place conditioning was obtained in LC and PF offspring when either 2.0 or 5.0 mg/kg of cocaine was paired with the designated place. In contrast, C40 offspring did not exhibit place conditioning at either training dose. Yet, all animals exposed to 5 mg/kg of cocaine during conditioning exhibited less activity during the test (when no cocaine was given) than controls given unpaired exposures to the apparatus and cocaine and C40 offspring did not differ from LC and PF offspring in this respect. Therefore, despite their lack of a conditioned place preference for cocaine, rats that had been exposed gestationally to cocaine nevertheless revealed an effect of cocaine during conditioning in one aspect of their test behavior. Possible explanations for the lack of cocaine-induced place preference in these animals include a learning deficit or a change in cocaines effectiveness as a reward.

Some parameters of conditioned immunosuppression: Species difference and CS-US delay

Three experiments were conducted in which an illness-inducing immunosuppressant, cyclophosphamide (an unconditioned stimulus, US) was associated with a previously presented saccharin solution conditioned stimulus (CS). In each experiment, reexposure to the CS produced a conditioned suppression of the plaque-forming-cell response in the experimental groups. Experiment I demonstrated this result with Fisher 344 rats. Experiment II replicated the effect with Balb/c mice. In Experiment III conditioned immunosuppression was demonstrated when mice received CS-US delays as long as 6 hours. No evidence of a delay gradient was present in either the behavioral or the immunologic data. These parallel findings offer no support for the idea of a dissociation between the taste aversion and conditioned immunosuppression processes.

Disruption of neophobia, conditioned odor aversion, and conditioned taste aversion in rats with hippocampal lesions.

Previous studies have implicated the hippocampus in the acquisition of conditioned taste aversions. However, the effect of hippocampal (HPC) lesions on the acquisition of conditioned aversions to the distal olfactory cue has not been investigated. In this study rats with bilateral electrolytic hippocampal lesions were given access to an odor conditioned stimulus (CS) alone or a compound odor-taste CS, followed by an injection of LiCl or saline. The results indicated that HPC lesions attenuated the neophobic response to both CSs, and disrupted conditioned odor and taste aversions, relative to sham-operated controls. Furthermore, the disruption in conditioned odor aversions could not be attributed to attenuation of neophobia in lesioned subjects nor to prolonged neophobia in sham-operated controls. The results are consistent with pharmacological studies in suggesting that the hippocampus is involved in the formation of conditioned odor aversions.

Opiate receptor supersensitivity produced by chronic naloxone treatment: dissociation of morphine-induced antinociception and conditioned taste aversion.

In three separate experiments, rats were used to assess the effects of chronic administration of naloxone on specific binding of 3H-naloxone in various regions of the central nervous system (CNS) and on the efficacy of morphine to produce antinociception and a conditioned taste aversion. Chronic naloxone treatment increased opiate binding in medulla-pons, midbrain, hypothalamus, hippocampus, striatum, and prefrontal cortex, but not in either spinal cord or cerebellum. In those CNS regions exhibiting increased opiate binding, the duration of increased binding following termination of the naloxone treatment differed between regions. In conjunction with the increase in opiate binding, the efficacy of morphine to produce antinociception was potentiated, while the efficacy to produce a conditioned taste aversion was unchanged. Moreover, the administration of naloxone during behavioral testing blocked completely the antinociceptive effect, but not the aversive effect, of morphine. These results indicate that morphine-induced antinociception and conditioned taste aversion may be dissociated neuropharmacologically.

Differential effectiveness of various prior-cuing treatments in the reactivation and maintenance of memory.

In 3 experiments, changes were examined in the characteristics of newly acquired and reinstated memories over time in preweanling rats. Experiment 1 indicated that forgetting after conditioning was monotonic, with the upper limit of retention at approximately 120 min posttraining. In Experiment 2, Ss were exposed to various elements of the training episode before testing, after either a 3- or a 24-hr retention interval. The results indicated that the prior-cuing treatments were differentially effective and that the effectiveness of a reactivation treatment may change as a function of the retention interval. Experiment 3 indicated that Ss expressed a conditioned aversion at much longer intervals following reactivation treatments than after initial conditioning. Furthermore the susceptibility of the reinstated memory to forgetting was dependent on the prior-cuing treatment used. The results suggest a change in the memorial representation of the conditioning episode over time.

Age-related differences in short-term retention of separable elements of an odor aversion.

The rate of forgetting over short intervals was tested in preweanling rats, 8, 12, or 18 days postnatal, using procedures that may have analytical advantages over other tests of short-term retention. Separate tests of retention were conducted for the simple occurrence of an odor and for the occurrence of an odor paired with a mild footshock. Forgetting of odors with either of two histories, incidental or target, was more rapid the younger the preweanling, over intervals of less than an hour. There was some indication of more rapid forgetting for incidental than target odors. Finally, although exposure to a CS- (an odor not paired with footshock) was necessary for conditioning of the CS+ (an odor paired with footshock) in rats 8 or 12 days of age, exposure to a CS- had no influence on conditioning of the CS+ in preweanlings 18 days of age. The age-related differences in forgetting over intervals less than an hour long suggest that substantial age-related differences in forgetting can occur that, it is likely, are not accounted for by differential growth.