Plasticity-related genes in brain development and amygdala-dependent learning.

Learning about motivationally important stimuli involves plasticity in the amygdala, a temporal lobe structure. Amygdala-dependent learning involves a growing number of plasticity-related signaling pathways also implicated in brain development, suggesting that learning-related signaling in juveniles may simultaneously influence development. Here, we review the pleiotropic functions in nervous system development and amygdala-dependent learning of a signaling pathway that includes brain-derived neurotrophic factor (BDNF), extracellular signaling-related kinases (ERKs) and cyclic AMP-response element binding protein (CREB). Using these canonical, plasticity-related genes as an example, we discuss the intersection of learning-related and developmental plasticity in the immature amygdala, when aversive and appetitive learning may influence the developmental trajectory of amygdala function. We propose that learning-dependent activation of BDNF, ERK and CREB signaling in the immature amygdala exaggerates and accelerates neural development, promoting amygdala excitability and environmental sensitivity later in life.

Altered brain development in an early-onset murine model of Alzheimer's disease.

Murine models of Alzheimer's disease (AD) have been used to draw associations between atrophy of neural tissue and underlying pathology. In this study, the early-onset TgCRND8 mouse model of AD and littermate controls were scanned longitudinally with in vivo manganese-enhanced MRI (MEMRI) before and after the onset of amyloid plaque deposition at 12 weeks of age. Separate cohorts of mice were scanned at 1 week (ex vivo imaging) and 4 weeks (MEMRI) of age to investigate early life alterations in the brain. Contrary to our expectations, differences in neuroanatomy were found in early post-natal life, preceding plaque deposition by as much as 11 weeks. Many of these differences remained at all imaging time points, suggesting that they were programmed early in life and were unaffected by the onset of pathology. Furthermore, rather than showing atrophy, many regions of the TgCRND8 brain grew at a faster rate compared to controls. These regions contained the greatest density of amyloid plaques and reactive astrocytes. Our findings suggest that pathological processes as well as an alteration in brain development influence the TgCRND8 neuroanatomy throughout the lifespan.

Emerging roles for MEF2 transcription factors in memory.

In the brain, transcription factors are critical for linking external stimuli to protein production, enabling neurons and neuronal networks to adapt to the ever-changing landscape. Gene transcription and protein synthesis are also vital for the formation of long-term memory. Members of the myocyte enhancer factor-2 (MEF2) family of transcription factors have a well-characterized role in the development of a variety of tissues, but their role in the adult brain is only beginning to be understood. Recent evidence indicates that MEF2 regulates the structural and synaptic plasticity underlying memory formation. However, in stark contrast to most other transcription factors implicated in memory, MEF2-mediated transcription constrains (rather than promotes) memory formation. Here, we review recent data examining the role of MEF2 in adult memory formation in rodents.

Inducible repression of CREB function disrupts amygdala-dependent memory.

Evidence from Aplysia, Drosophila, mice, and rats indicates that the CREB (cAMP/Ca2+ responsive element binding protein) family of transcription factors is critical for long-term memory. Recent findings, however, suggest that performance abnormalities may contribute to the memory deficits attributed to CREB manipulations in mammals. To clarify the role of CREB in memory, we used a paradigm, conditioned taste avoidance, that places few performance demands on the subject. We show that lesioning or blocking protein synthesis in the basolateral amygdala of mice disrupts conditioned taste aversion. Furthermore, either chronically or acutely disrupting CREB function in two different types of genetically modified mice blocks memory for conditioned taste aversion measured 24 h following training. Together, these findings indicate that CREB-mediated transcription and protein synthesis are required for conditioned taste aversion memory.

Long-term memory is facilitated by cAMP response element-binding protein overexpression in the amygdala.

At least two temporally and mechanistically distinct forms of memory are conserved across many species: short-term memory that persists minutes to hours after training and long-term memory (LTM) that persists days or longer. In general, repeated training trials presented with intervening rest intervals (spaced training) is more effective than massed training (the same number of training trials presented with no or short intervening rest intervals) in producing LTM. LTM requires de novo protein synthesis, and cAMP response element-binding protein (CREB) may be one of the transcription factors regulating the synthesis of new proteins necessary for the formation of LTM. Here we show that rats given massed fear conditioning training show no or weak LTM, as measured by fear-potentiated startle, compared with rats given the same amount of training but presented in a spaced manner. Increasing CREB levels specifically in the basolateral amygdala via viral vector-mediated gene transfer significantly increases LTM after massed fear training. The enhancing effect of CREB overexpression on LTM formation is shown to be specific in terms of biochemistry, anatomy, time course, and the training procedure used. These results suggest that CREB activity in the amygdala serves as a molecular switch for the formation of LTM in fear conditioning.

Computer-assisted behavioral assessment of Pavlovian fear conditioning in mice.

In Pavlovian fear conditioning, a conditional stimulus (CS, usually a tone) is paired with an aversive unconditional stimulus (US, usually a foot shock) in a novel context. After even a single pairing, the animal comes to exhibit a long-lasting fear to the CS and the conditioning context, which can be measured as freezing, an adaptive defense reaction in mice. Both context and tone conditioning depend on the integrity of the amygdala, and context conditioning further depends on the hippocampus. The reliability and efficiency of the fear conditioning assay makes it an excellent candidate for the screening of learning and memory deficits in mutant mice. One obstacle is that freezing in mice has been accurately quantified only by human observers, using a tedious method that can be subject to bias. In the present study we generated a simple, high-speed, and highly accurate algorithm that scores freezing of four mice simultaneously using NIH Image on an ordinary Macintosh computer. The algorithm yielded a high correlation and excellent linear fit between computer and human scores across a broad range of conditions. This included the ability to score low pretraining baseline scores and accurately mimic the effects of two independent variables (shock intensity and test modality) on fear. Because we used a computer and digital video, we were able to acquire a secondary index of fear, activity suppression, as well as baseline activity scores. Moreover, we measured the unconditional response to shock. These additional measures can enhance the sensitivity of the assay to detect interesting memory phenotypes and control for possible confounds. Thus, this computer-assisted system for measuring behavior during fear conditioning allows for the standardized and carefully controlled assessment of multiple aspects of the fear conditioning experience.

Behavioral effects of clozapine and dopamine receptor subtypes.

The atypical neuroleptic clozapine (CLZ) is an extremely effective antipsychotic that produces relatively few motoric side effects. However, CLZ displays limited antagonism at the dopamine (DA) D2 receptor, the receptor commonly thought to mediate the antipsychotic activity of neuroleptics. The mechanism of action behind the efficacy of CLZ remains to be determined. Miller, Wickens and Beninger [Progr. Neurobiol., 34, 143-184 (1990)] propose a "D1 hypothesis of antipsychotic action" that may explain the antipsychotic effects of CLZ. This hypothesis is built on the interactions between D2, cholinergic and D1 mechanisms in the striatum. These authors assert that although typical neuroleptics block D2 receptors, it is through an indirect action on D1 receptors that their antipsychotic action is manifest. The extra-pyramidal side effects produced by typical neuroleptics are hypothesized to be due to an indirect action on cholinergic receptors. It is argued that the anticholinergic properties of CLZ negate the D2 (motor side effects) action of CLZ, allowing CLZ to diminish psychotic symptoms through a direct action on D1 receptors. Thus, CLZ may function as a D1 receptor antagonist in behavioral paradigms. The current paper reviews and compares the behavioral profile of CLZ to those produced by D2- and D1-selective antagonists with specific reference to unconditioned and conditioned behaviors in order to more fully evaluate the "D1 hypothesis of CLZ action". Although the actions of CLZ remain unique, they do share some striking similarities with D1 receptor antagonists especially in tests of unconditioned behavior, possibly implicating the D1 receptor in the action of this antipsychotic drug.

Evidence for CCK(A) receptor involvement in the acquisition of conditioned activity produced by cocaine in rats.

Cholecystokinin (CCK) is co-localized with dopamine (DA) in the nucleus accumbens (NAC) where evidence suggests that CCK(B) receptor-mediated mechanisms inhibit, while CCK(A) receptor-mediated mechanisms facilitate, DA function. As DA has been implicated in the acquisition of conditioned activity, the present experiments investigated the effects of CCK(A) and CCK(B) receptor selective antagonists on the acquisition and expression of conditioned activity produced by cocaine. Paired rats received four cocaine-environment pairings whereas Unpaired rats received the two stimuli explicitly unpaired, in that they received cocaine in the home cage. Using this procedure, cocaine (10 mg/kg, i.p.) reliably produced conditioned activity such that the Paired group showed a higher level of locomotion than the Unpaired group on a subsequent drug-free test day. Systemic administration of devazepide, a CCK(A) receptor antagonist, but not L-365,260, a CCK(B) receptor antagonist, blocked the acquisition of conditioned activity. Microinjection of the CCK(A) antagonist PD-140548 into the NAC similarly impaired the acquisition of conditioned activity. However, systemic administration of neither the CCK(A) nor CCK(B) receptor antagonist modified the expression of cocaine-induced conditioned activity once established. These studies suggest that CCK(A), but not CCK(B), receptor mediated mechanisms in the NAC play a key role in the acquisition of conditioned activity.

Activation of amygdala cholecystokininB receptors potentiates the acoustic startle response in the rat.

The acoustic startle reflex is a sensitive index of "anxiety" and "fear." Potentiation of startle by conditioned and unconditioned fear stimuli appears to be mediated by the amygdala. CholecystokininB (CCKB) agonists increase "anxiety" in laboratory animals and induce "panic" in humans. Here, we investigate the role CCKB receptor-mediated mechanisms in the amygdala in the potentiation of startle. First, intra-amygdala infusions of the CCKB receptor agonist pentagastrin (0, 0.01, 0.1, 1, and 10 nM) produced a dose-related potentiation of acoustic startle responses. At the highest dose, startle amplitudes were increased up to 90% above preinfusion baseline levels. Second, similar infusions of pentagastrin had no effect on locomotor activity over the same time course, showing that increases in startle responsivity after infusions of pentagastrin are not attributable to nonspecific changes in motor activity. Third, infusions of similar doses of pentagastrin into the striatum or nucleus accumbens did not potentiate startle responses. Fourth, pretreatment with the CCKB receptor antagonist L-365,260 (0.1 mg/kg, i.p.) attenuated the potentiation of startle produced by intra-amygdala infusions of pentagastrin. Finally, intra-amygdala infusion of the CCKB receptor-selective antagonist PD-135158 (10 micro;g) blocked the potentiation of startle produced by i.c.v. infusions of pentagastrin, suggesting that i.c.v. infusions of pentagastrin potentiate startle responses via activation of amygdala CCKB receptors. These results show that amygdala CCKB receptor-mediated mechanisms are involved in the potentiation of acoustic startle responses.

Opposing effects of CCK(A) and CCK(B) antagonists on the development of conditioned activity in rats.

Cholecystokinin (CCK) is co-localized with dopamine (DA) in portions of the mesolimbic system. Evidence suggests that CCK(B) receptor mechanisms mediate an inhibitory effect on DA function while CCK(A) receptor mechanisms mediate a facilitory effect on DA function. As DA has been implicated in the acquisition of conditioned activity, the present experiments investigated the interaction between selective CCK(A) and CCK(B) receptor antagonists on the development of conditioned activity. Experiment 1 determined whether the CCK(B) receptor antagonist, L-365,260 (0.01, 0.1 and 1.0mg/kg, i.p.) would induce conditioned activity. Paired groups received four two-hour drug-environment pairings whereas Unpaired groups received the two stimuli explicitly unpaired. The group that received L-365,260 (0.mg/kg) paired with the test environment subsequently showed increased activity in that environment in a drug-free test day. Experiment 2 examined the interaction of pre-treatment with the CCK(A) receptor antagonist, devazepide (0, 0.001, 0.01 and 0.1mg/kg) on the conditioned activity induced by L-365,260 (0.1mg/kg). Devazepide impaired the acquisition of L-365,260 conditioned activity. These studies suggest that CCK mechanisms play a key role in the development of conditioned activity and extend previous research suggesting a bi-directional function for CCK(A) and CCK(B) receptors.

Acquisition of conditioned reward blocked by intra-accumbens infusion of PD-140548, a CCKA receptor antagonist.

Cholecystokinin (CCK) is an endogenous peptide that is colocalized with dopamine (DA) in some mesolimbic neurons projecting to the nucleus accumbens (NAC). DA has been implicated in the acquisition of conditioned rewarding properties by neutral stimuli [conditioned stimuli (CS)] associated with a primary reward (such as food). A variety of experimental evidence suggests that exogenously applied CCK, acting at the CCKA receptor, potentiates the function of DA in the NAC. Thus, the present experiment examined the role of endogenous CCKA mechanisms in the NAC in the development of conditioned reward. The CCKA receptor-selective antagonist PD-140548 was microinjected into the NAC during the CS-food pairing phase of a conditioned reward experiment. In the test session, animals that previously received vehicle microinjections into the NAC or PD-140548 microinjections into areas surrounding the NAC pressed a lever that produced the CS more often than did a control lever. The CS had gained conditioned rewarding properties. However, animals that received PD-140548 microinjections into the NAC did not exhibit a preference for the CR lever. Results suggest that blockade of CCKA receptors in the NAC impairs the development of conditioned reward. These findings support a role for endogenous CCKA mechanisms in the NAC in the acquisition of stimulus-reward associations.

Intracerebroventricular infusion of the CCKB receptor agonist pentagastrin potentiates acoustic startle.

The acoustic startle reflex is increased by stimuli associated with aversive events (such as the delivery of shock) and so has been used as a sensitive index of 'anxiety' or 'fear'. Administration of cholecystokininB (CCKB) receptor agonists produces a constellation of behaviors associated with 'anxiety' in laboratory animals and humans. Here, intracerebroventricular infusions of the CCKB agonist, pentagastrin (0, 1, 10, 100 nM), produced a long-lasting, dose-related potentiation of acoustic startle responses. Similar infusions of pentagastrin had no effect on locomotor activity over the same time course, showing that changes in startle responses following infusions of pentagastrin are not due to nonspecific changes in motor activity.

PD-135158, a CCKB receptor antagonist, microinjected into the nucleus accumbens and the expression of conditioned rewarded behavior.

Cholecystokinin (CCK) has been localized in the nucleus accumbens (NAC) where it may interact with dopamine neurotransmission. NAC dopamine is involved in the control over behavior produced by conditioned rewards. The present experiment examines whether the blockade of CCKB receptors in the NAC with microinjection of PD-135158 (10 micrograms in 0.5 microliter) potentiates bar-pressing for stimuli previously associated with food reward. Intra-NAC microinjections of amphetamine (10 micrograms in 0.5 microliter) increased the number of bar presses for conditioned reward presentation. Furthermore, similar administration of PD-135158 produced no significant effect on responding when administered alone but potentiated the level of amphetamine responding. These findings suggest that endogenous CCKB mechanisms in the NAC may normally inhibit dopamine function in reward-related behaviors.

Devazepide, a CCKA receptor antagonist, impairs the acquisition of conditioned reward and conditioned activity.

Cholecystokinin (CCK) is co-localized with dopamine (DA) in portions of the mesolimbic system, where it may facilitate the function of DA through the CCKA receptor subtype. DA has been implicated in the acquisition of conditioned incentive learning, raising the possibility of a role for endogenous CCK in this learning process. This hypothesis was tested using two complementary behavioral paradigms. Experiment 1 examined the effects of systemic administration of the CCKA receptor selective antagonist, devazepide (0, 0.001, 0.01, 0.1 mg/kg), on the acquisition of conditioned reward. Two novel levers were presented to drug-free animals in a test session; depression of the conditioned reward (CR) lever produced a light-tone stimulus previously paired with food availability while depression of the non-CR lever produced no programmed consequence. Animals receiving vehicle pretreatment in the food-CS conditioning sessions responded more frequently on the CR lever during the test session. However, pre-treatment with devazepide (0.1 mg/kg but not 0.001 or 0.01 mg/kg) in the conditioning sessions blocked the acquisition of conditioned reward. In contrast, experiment 2 showed that the development of conditioned reward was not affected by similar administration of the CCKB selective antagonist, L-365,260 (0, 0.001, 0.01, or 0.1 mg/kg). The possibilities that devazepide (0.1 mg/kg) impaired the development of conditioned reward by decreasing the amount of food consumed or by inducing a conditioned taste aversion to the food were ruled out in experiments 3 and 4. The effects of devazepide on the acquisition of conditioned activity induced by amphetamine were assessed in experiment 5. During four conditioning sessions, rats received devazepide (0, 0.001, 0.01, 0.1 or 1.0 mg/kg) treatment prior to amphetamine-environment pairings. The conditioned activity effect was demonstrated if on the subsequent drug-free test day the environment alone elicited increased locomotion. Devazepide (0.1 or 1.0 mg/kg) attenuated the development of conditioned activity. Together, these results provide converging evidence that intact CCKA function may be necessary for the development of conditioned incentive learning.

The CCKB antagonist, L-365,260, attenuates fear-potentiated startle.

The neuropeptide cholecystokinin (CCK), via the CCKB receptor, increases behaviors associated with anxiety in laboratory animals and humans. The present experiment assessed the role of endogenous CCKB function in fear-potentiated startle, a test of "anxiety" in rats. The amplitude of the acoustic startle response is potentiated if preceded by a stimulus that has been previously paired with shock. Pretreatment with the CCKB antagonist L-365,260 (0, 0.1, 1.0, and 10.0 mg/kg, IP) did not affect baseline acoustic startle amplitudes, but dose-dependently decreased fear-potentiated startle. These results indicate that the specific attenuation of fear-potentiated startle induced by L-365,260 was not due to a general decrease in motor responsivity. The present findings are consistent with the effects of CCKB antagonists in other tests measuring anxiety in animals.

Interaction of CCKB receptors with amphetamine in responding for conditioned rewards.

Cholecystokinin (CCK) has been localized in the nucleus accumbens (NAC) where it may interact with dopamine neurotransmission. NAC dopamine is involved in the control over behavior produced by conditioned rewards. The present experiment tested the whether blockade of endogenous CCKB receptors with L-365,260 (0.1 mg/kg, IP) potentiates bar pressing for stimuli previously associated with food reward. Intra-accumbens amphetamine (20 micrograms) facilitated bar pressing for conditioned rewards. Systemic administration of L-365,260 potentiated this amphetamine response but produced no effect on responding when administered alone. These findings suggest that endogenous CCKB mechanisms may normally inhibit DA function in reward-related behaviors.

Neuropeptide Y: intraaccumbens injections produce a place preference that is blocked by cis-flupenthixol.

Neuropeptide Y (NPY) has been localized in the nucleus accumbens (NAcc), where it may influence dopamine (DA) neurotransmission. Extensive data implicate NAcc DA in reward-related learning, raising the possibility that NPY microinjected into the NAcc may induce rewarding effects mediated by DA. This hypothesis was tested using the conditioned place preference (CPP) paradigm. Each experiment consisted of three distinct phase: preconditioning (three 15-min exposures to an apparatus with two compartments connected by a tunnel); conditioning (four 30-min pairing of one compartment with drug and four similar pairings of the other compartment with vehicle); and test (three 15-min exposures to the apparatus). A significant increase in the time spent in the drug-paired compartment from preconditioning to test was taken as evidence of a CPP. Two experiments showed that systemic (2.0 mg/kg, IP) or intraaccumbens amphetamine (10.0 micrograms in 0.5 microliters on each side) produced a CPP. The third experiment showed that intraaccumbens NPY (0.1 micrograms in 0.5 microliter on each side) produced a CPP. This CPP was blocked by pretreatment with a dose of the DA receptor blocker cis-flupenthixol (20.0 micrograms in 0.5 microliter on each side in the NAcc) that, alone, produced no CPP effect. These results strongly suggest that NPY applied to the NAcc is rewarding. In addition, these rewarding properties of NPY may be mediated by DA neurotransmission.

Behavioral effects of intrastriatal caffeine mediated by adenosinergic modulation of dopamine.

Although caffeine is generally classified as a psychomotor stimulant, the neurotransmitter systems mediating its effect on behavior have not yet been established. Mounting evidence suggests possible involvement of adenosinergic and/or dopaminergic (DA) systems. To evaluate these possibilities, four experiments examined circling behavior in rats following unilateral intrastriatal microinjections of: 1) caffeine alone; 2) the adenosine agonist, 2-chloroadenosine (2-CADO) alone; 3) caffeine with 2-CADO pretreatment; and 4) caffeine with pretreatment of the DA receptor antagonist, cis-flupenthixol. Each experiment consisted of seven test sessions; the first and seventh were preceded by no treatment, the second and sixth by control microinjections (saline or cis-flupenthixol) and the third, fourth and fifth by drug microinjections. Results showed that 10.0 and 20.0 but not 1.0 micrograms of caffeine produced a significant contraversive bias in circling behavior, while 2.0 and 5.0 but not 1.0 microgram doses of 2-CADO produced significant ipsiversive circling. Rats pretreated with central 2-CADO or cis-flupenthixol (in doses that did not influence circling bias when administered alone) prior to caffeine (10.0 micrograms) failed to exhibit a contraversive bias. Taken together, the present studies provide compelling support for the suggestion that the motor effects of intrastriatal caffeine are mediated by the antagonism of endogenous adenosine which, in turn, functionally increases DA.