Anthony G. Romano

Retention and acquisition of classical trace conditioned responses by rabbits with hippocampal lesions.

The effects of dorsal hippocampal lesions on retention of classical trace conditioned responses were examined using the rabbit nictitating membrane preparation. Animals were trained to criteria and then lesioned either in the cortex or in the hippocampus and the cortex. Hippocampal damage had no effect on the retention of responses but produced significantly longer onset latencies. A control group of hippocampal animals acquired conditioned responses (CRs) at least as quickly as the prelesion subjects, and they also exhibited longer response onset latency. A second experiment evaluated the performance of hippocampal lesioned animals in classical trace conditioning with either a low-intensity periorbital shock or corneal air puff as the unconditioned stimulus (UCS). Hippocampal animals successfully acquired CRs under both conditions but exhibited an alteration of response onset which was dependent on the form of the UCS. Hippocampal animals displayed shorter response onset in the air-puff condition and longer response onset in the shock condition. Cortical animals timed responses consistently regardless of the UCS. These findings strongly suggest that the hippocampus modulates temporal characteristics of learned behavior.

Prenatal exposure to cocaine disrupts discrimination learning in adult rabbits

Previous studies had shown that intrauterine exposure to cocaine produces an increase in the number of immunoreactive GABA neurons and abnormal dendritic structure of pyramidal cells in the anterior cingulate cortex, a brain region known to be involved in attentional processes and discrimination learning. Because structural abnormalities might be expected to produce related functional deficits, we examined whether intrauterine exposure to cocaine would affect discrimination learning in adult rabbits. We previously reported that cocaine progeny undergoing concurrent acquisition to visual and auditory CSs show a normal rate of learning to a light CS and an accelerated rate of learning to a tone. Here, we report that adult, Dutch-belted rabbits exposed to cocaine in utero showed impaired discrimination learning when responding to a positive visual cue but not when responding to a positive auditory cue. The nature of the deficit consisted of an impaired ability to acquire learned responses to the visual CS+ rather than in an impaired ability to withhold responses to the auditory CS-. Given that auditory stimuli tend to be more salient than visual stimuli in the normal rabbit, the preceding pattern of results suggests that intrauterine cocaine exposure affected the ability to preferentially attend to less salient but relevant stimuli and to ignore more salient, irrelevant stimuli. More importantly, these results indicate that prenatal exposure to cocaine produces neurobehavioral abnormalities which persist into adult life.

Effects of serotonin 5-HT(2A/2C) antagonists on associative learning in the rabbit.

Abstract The 5-HT2A/2C receptor antagonist, ritanserin, was reported to retard the acquisition of conditioned responses (CRs) during classical conditioning of the rabbit’s nictitating membrane (NM) response. The present study compared the effects of ritanserin on acquisition of CRs to a tone conditioned stimulus (CS) with that of the 5-HT2A/2C receptor antagonist, LY-53,857 and the 5-HT2A selective antagonist, MDL-11,939. All three drugs were injected at equimolar doses of 0.067, 0.67 and 6.7 μmol/kg, SC, 1 h before behavioral testing. Ritanserin and MDL-11,939 retarded CR acquisition to a tone CS, while LY-53,857 had no effect. Control experiments demonstrated that ritanserin (1 μmol/kg), MDL-11,939 (1 μmol/kg) and LY-53,857 (2 μmol/kg) had no effect on baseline responding or non-associative responding to the CS. However, both ritanserin and MDL-11,939 impaired the performance of the unconditioned NM reflex, as measured by a decrease in UR amplitudes on US alone trials, while LY-53,857 had no effect. In previously trained animals, ritanserin robustly impaired the performance of CRs, as measured by a reduced ability of the CS to elicit CRs, while the effects of LY-53,857 and MDL-11,939 were marginal. The retardation of associative learning produced by ritanserin and MDL-11,939 may have been due, at least in part, to their impairment of the NM reflex arc. Since MDL-11,939 is a highly selective 5-HT2A antagonist, the retardation of learning and impairment of UR amplitudes produced by MDL-11,939 and ritanserin may have been due to blockade of the 5-HT2A receptor. The ability of ritanserin and MDL-11,939 to produce effects on learning and performance that were opposite to that of 5-HT2A/2C agonists suggests that they may be acting as inverse agonists at that receptor. These results stress the importance of the serotonergic system for optimal associative learning and motor function.

Intrauterine exposure to cocaine produces a modality-specific acceleration of classical conditioning in adult rabbits

Previous studies had demonstrated that in utero exposure to cocaine produces structural changes in the development of the rabbits anterior cingulate cortex. Because the anterior cingulate cortex has been proposed to subserve a variety of cognitive processes including associative learning, we investigated the effects of intrauterine exposure to cocaine on the acquisition of the rabbits classically conditioned nictitating membrane response. Adult, sexually mature rabbits born of dams that had received intravenous injections of either saline or cocaine (4 mg/kg, twice a day) from day 8 to day 29 of gestation were classically conditioned by pairing tone and light CSs with an airpuff US. Rabbits that had been exposed to cocaine in utero demonstrated a more rapid acquisition of CRs to a tone CS but not to a light CS as compared with saline controls. Control experiments indicated that the accelerated learning to the tone CS was not due to sensitization, pseudoconditioning, altered baseline rate of responding, an increased responsiveness to the airpuff US, or to a change in the intensity threshold of the tone CS for elicitation of CRs. We conclude that in utero exposure to cocaine alters the processing of auditory stimuli and this leads to an abnormally rapid acquisition of CRs. It is suggested that this functional consequence of prenatal exposure to cocaine is due to structural abnormalities in anterior cingulate cortex.

Effects of prenatal exposure to cocaine on the developing brain: Anatomical, chemical, physiological and behavioral consequences

Earlier studies of human infants and studies employing animal models had indicated that prenatal exposure to cocaine produced developmental changes in the behavior of the offspring. The present paper reports on the results obtained in a rabbit model ofin utero exposure to cocaine using intravenous injections (4 mg/kg, twice daily) that mimic the pharmacokinetics of crack cocaine in humans. At this dose, cocaine had no effect on the body weight gain of dams, time to delivery, litter size and body weight or other physical characteristics of the offspring. In spite of an otherwise normal appearance, cocaine-exposed neonates displayed a permanent impairment in signal transduction via the D1 dopamine receptor in caudate nucleus, frontal cortex and cingulate cortex due to an uncoupling of the receptor from its associated Gs protein. This uncoupling in the caudate nucleus was shown to have behavioral consequences in that young or adult rabbits, exposed to cocainein utero, failed to demonstrate amphetamine-elicited motor responses normally seen after activation of D1 receptors in the caudate. The cocaine progeny also demonstrated permanent morphological abnormalities in the anterior cingulate cortex due to uncoupling of the D1 receptor and the consequent inability of dopamine to regulate neurite outgrowth during neuronal development. Consistent with the known functions of the anterior cingulate cortex, adult cocaine progeny demonstrated deficits in attentional processes. This was reflected by impairment in discrimination learning during classical conditioning that was due to an inability to ignore salient stimuli even when these were not relevant to the task. The impairment in discrimination learning also occurred in an instrumental avoidance task and could be shown to be due to an impairment of cingulothalamic learning-related neuronal coding. It was proposed that the selective loss of D1-related neurotransmission in the anterior cingulate cortex prevented an appropriate activation of GABA neurons and thus a loss of inhibitory regulation that is necessary for processes involved in associative attention. Taken together, these findings suggest that the uncoupling of the D1 receptor from its G protein may be the fundamental source of the anatomic, cognitive and motor disturbances seen in rabbits exposed to cocainein utero. Moreover, the long-term cognitive and motor deficits observed in the rabbit model are in agreement with recent reports indicating that persistent attentional and other behavioral deficits may be evident in cocaine-exposed children as they grow older and are challenged to master more complex cognitive tasks.

Prenatal Cocaine Exposure: Long-Term Deficits in Learning and Motor Performancea

ABSTRACT: We have developed a rabbit model of in utero exposure to intravenous injections of cocaine given twice daily to dams from gestational days 8–29. At the doses employed (4 mg/kg, injected twice daily), no differences were found in the body weight gain of dams, time to delivery, litter size, and body weight or other physical characteristics of the offspring. However, cocaine‐exposed pups displayed an abnormal structural and neurochemical development of the anterior cingulate cortex which persisted into adulthood. In agreement with the known functions of the anterior cingulate cortex, we found that adult, sexually mature rabbits, exposed to cocaine prenatally, demonstrate impairments in motor function, alterations in associative learning and severe impairments in discrimination learning. Moreover, the alterations in discrimination learning were interpreted to be due to deficits in attentional processes. Specifically, cocaine progeny preferentially attend to more salient stimuli even when these are not relevant to the task. Consequently they have difficulty in attending to less salient but relevant stimuli when more salient but irrelevant stimuli occur in the same context. We concluded that the learning deficits are a reflection of the morphologic and neurochemical abnormalities of the anterior cingulate cortex. Alterations in dopamine function of the caudate nucleus may also contribute to the deficits in motor performance.

Effects of LSD, Ritanserin, 8-OH-DPAT, and Lisuride on Classical Conditioning in the Rabbit

d-Lysergic acid diethylamide (LSD), an agonist at the 5-HT(2A/2C) and 5-HT1A receptors, has previously been demonstrated to enhance associative learning as measured by accelerated acquisition of the rabbits classically conditioned nictitating membrane (NM) response. The present study examined further the role of these receptors in the action of LSD. LSD (30 nmol/kg, I.V.) significantly enhanced conditioned response (CR) acquisition to both tone and light conditioned stimuli (CSs), while the 5-HT1A receptor agonists 8-hydroxy-2-(dipropylamino)tetralin (8-OH-DPAT; 50 and 200 nmol/kg) and lisuride (0.3-30 nmol/kg) had no effect. Ritanserin (6.7-6700 nmol/kg, S.C.), a selective 5-HT(2A/2C) receptor antagonist, retarded acquisition of CRs to both tone and light CSs in a dose-dependent manner. Ritanserin (6.7-670 nmol/kg, S.C.) also dose dependently antagonized the enhancement of CR conditioning produced by LSD (30 nmol/kg, I.V.) to both tone and light CSs. We conclude that the enhancement of CR acquisition by LSD was due to an action at the 5-HT(2A/2C) receptor. These results suggest that the 5-HT(2A/2C) receptor plays an important role in learning.

Selective remodeling of rabbit frontal cortex: relationship between 5-HT2A receptor density and associative learning

RationaleAssociative learning during classical trace eyeblink conditioning has been shown to be regulated by serotonin 5-HT2A receptors and to be critically dependent on the integrity of frontal cortex. Chronic administration of 5-HT2A ligands has been shown to produce a selective up- or down-regulation of 5-HT2A receptors in frontal cortex.ObjectivesWe examined whether alterations in 5-HT2A receptor density had a functional significance with respect to associative learning.MethodsAnimals received chronic injections of LSD, BOL or MDL11,939 and 1 day later began classical trace conditioning of the eyeblink response.ResultsThe density of 5-HT2A receptors in frontal cortex was significantly increased at 1–4 days after the cessation of chronic injections of the selective 5-HT2A receptor ligand MDL11,939. Rabbits demonstrated an enhancement of associative learning when training began at 1 day after cessation of chronic MDL11,939 injections, but acquired at the same rate as controls when training began at 8 days after cessation of injections, a time when receptor density had returned to control levels. Animals that began training 1 day after chronic injections of BOL or LSD, drugs that produce decreases in 5-HT2A receptor density, demonstrated normal rates of acquisition.ConclusionsThese results indicate that increases in the density of 5-HT2A receptors in frontal cortex are associated with increases in the rate of associative learning, and further support an important role for this receptor in cortical circuitry that mediates learning. More generally, these results suggest an approach for functional remodeling of brain regions in the adult animal.

Effect of 5-HT2 receptor antagonists on a cranial nerve reflex in the rabbit: evidence for inverse agonism

Abstract This study examined the role of the serotonin 5-HT2 receptor in motor function by examining the effect of antagonists on the motor performance of a cranial nerve reflex, the nictitating membrane (NM) reflex of the rabbit. The NM reflex was elicited by varying intensities of a tactile stimulus and the magnitudes of the elicited responses were measured at each intensity. Dose-response curves were obtained for the effects of several 5-HT2 receptor antagonists on response magnitude. d-Bromolysergic acid diethylamide (BOL), LY-53,857 and ketanserin had no significant effect on the magnitude of the NM reflex, indicating that they are neutral antagonists. However, the 5-HT2 receptor antagonists ritanserin, MDL-11,939 and mianserin produced a significant reduction in response magnitude with no significant effects on response frequency, suggesting that they were acting as inverse agonists at the 5-HT2 receptor. The reduction in reflex magnitude produced by mianserin (10 µmol/kg) was fully blocked by BOL (5.8 µmol/kg), supporting the conclusion that mianserin was producing a reduction in reflex magnitude through an effect at the 5-HT2 receptor. The occurrence of inverse agonism suggests the possible existence of constitutive activity in vivo. We conclude that the 5-HT2 receptor (either 2A or 2C) plays an important role in motor function, perhaps by providing a tonic influence on motor systems.

A 5-HT2C agonist elicits hyperactivity and oral dyskinesia with hypophagia in rabbits

Serotonergic 5-HT2C and 5-HT1B receptors mediate inhibitory controls of eating. Questions have arisen about potential behavioral and neurological toxicity of drugs that stimulate the 2C site. We evaluated eating and other motor responses in male Dutch-belted rabbits after administration of m-chlorophenylpiperazine (mCPP). Studies conducted in vitro and in vivo assessed the pharmacological specificity of the ingestive actions of this agent. mCPP (0.15-10 micromol/kg sc) reduced consumption of chow and 20% sucrose solution with equal potencies (ED50 approximately equal 0.6 micromol/kg). In radioligand binding to rabbit cortex, mCPP displayed 15-fold higher affinity for 5-HT2C than for 5-HT1B receptors. The serotonin antagonist mesulergine (7000-fold selective for 5-HT2C) reversed the hypophagic action of mCPP, but the 5-HT1B/1D antagonist GR127,935 did not. GR127,935 (0.5 micromol/kg) did prevent hypophagia produced by the highly selective 5-HT1B/1D agonist GR46,611. Observational methods demonstrated that mCPP decreased the frequency of eating chow but increased other motor activities. When rabbits consumed sucrose, videoanalysis revealed that mCPP reduced total time licking and the duration of individual bouts, but not bout frequency or the actual rate of consumption. mCPP increased locomotor and other activities, and greatly increased vacuous oromotor stereotypies and tongue protrusions. Nonetheless, rabbits licked accurately at the spout for sucrose. When sucrose was infused intraorally through a cheek catheter, mCPP actually increased the peak amplitude and overall magnitude of jaw movements. We conclude that mCPP stimulates 5-HT2C receptors to reduce food intake in rabbits. This hypophagia involves disruption of appetitive components of eating and is accompanied by adverse motor actions. This profile raises questions about the use of the 5-HT2C receptor as a target for novel therapeutic agents for obesity.