A mouse model for the learning and memory deficits associated with neurofibromatosis type I.

Neurofibromatosis type I (NF1) is one of the most commonly inherited neurological disorders in humans, affecting approximately one in 4,000 individuals. NF1 results in a complex cluster of developmental and tumour syndromes that include benign neurofibromas, hyperpigmentation of melanocytes and hamartomas of the iris. Some NF1 patients may also show neurologic lesions, such as optic pathway gliomas, dural ectasia and aqueduct stenosis. Importantly, learning disabilities occur in 30% to 45% of patients with NF1, even in the absence of any apparent neural pathology. The learning disabilities may include a depression in mean IQ scores, visuoperceptual problems and impairments in spatial cognitive abilities. Spatial learning has been assessed with a variety of cognitive tasks and the most consistent spatial learning deficits have been observed with the Judgement of Line Orientation test. It is important to note that some of these deficits could be secondary to developmental abnormalities and other neurological problems, such as poor motor coordination and attentional deficits. Previous studies have suggested a role for neurofibromin in brain function. First, the expression of the Nf1 gene is largely restricted to neuronal tissues in the adult. Second, this GTPase activating protein may act as a negative regulator of neurotrophin-mediated signalling. Third, immunohistochemical studies suggest that activation of astrocytes may be common in the brain of NF1 patients. Here, we show that the Nf1+/- mutation also affects learning and memory in mice. As in humans, the learning and memory deficits of the Nf1+/- mice are restricted to specific types of learning, they are not fully penetrant, they can be compensated for with extended training, and they do not involve deficits in simple associative learning.

Inducible, pharmacogenetic approaches to the study of learning and memory.

Here we introduce a strategy in which pharmacology is used to induce the effects of recessive mutations. For example, mice heterozygous for a null mutation of the K-ras gene (K-ras+/-) show normal hippocampal mitogen-activated protein kinase (MAPK) activation, long-term potentiation (LTP) and contextual conditioning. However, a dose of a mitogen-activated/extracellular-signal-regulated kinase (MEK) inhibitor, ineffective in wild-type controls, blocks MAPK activation, LTP and contextual learning in K-ras+/- mutants. These indicate that K-Ras/MEK/MAPK signaling is critical in synaptic and behavioral plasticity. A subthreshold dose of NMDA receptor antagonists triggered a contextual learning deficit in mice heterozygous for a point mutation (T286A) in the alphaCaMKII gene, but not in K-ras+/- mutants, demonstrating the specificity of the synergistic interaction between the MEK inhibitor and the K-ras+/- mutation. This pharmacogenetic approach combines the high temporal specificity that pharmacological manipulations offer, with the molecular specificity of genetic disruptions.

Spaced training induces normal long-term memory in CREB mutant mice.

BACKGROUND: The cAMP responsive element binding protein (CREB) is a transcription factor the activity of which is modulated by increases in the intracellular levels of cAMP and calcium. Results from studies with Aplysia, Drosophila and mice indicate that CREB-activated transcription is required for long-term memory. Furthermore, a recent study found that long-term memory for olfactory conditioning can be induced with a single trial in transgenic Drosophila expressing a CREB activator, whereas in normal flies, with presumably lower CREB-mediated transcription levels, conditioning requires multiple spaced trials. This suggests that CREB-mediated transcription is important in determining the type of training required for long-term memory of olfactory conditioning in Drosophila. Interestingly, studies with cultured Aplysia neurons indicated that removing a CREB repressor promoted the formation of long-term facilitation, a cellular model of non-associative memory. RESULTS: Here, we have confirmed that mice lacking the alpha and Delta CREB proteins (CREBalphaDelta-) have abnormal long-term, but not short-term, memory, as tested in an ethologically meaningful task. Importantly, additional spaced training can overcome the profound memory deficits of CREBalphaDelta- mutants. Increasing the intertrial interval from 1 to 60 minutes overcame the memory deficits of the CREBalphaDelta- mice in three distinct behavioral tasks: contextual fear conditioning, spatial learning and socially transmitted food preferences. CONCLUSIONS: Previous findings and results presented here demonstrate that CREB mutant mice have profound long-term memory deficits. Importantly, our findings indicate that manipulations of CREB function can affect the number of trials and the intertrial interval required for committing information to long-term memory. Remarkably, this effect of CREB function is not restricted to simple conditioning tasks, but also affects complex behaviours such as spatial memory and memory for socially transmitted food preferences.

Impaired learning in mice with abnormal short-lived plasticity.

BACKGROUND: Many studies suggest that long term potentiation (LTP) has a role in learning and memory. In contrast, little is known about the function of short-lived plasticity (SLP). Modeling results suggested that SLP could be responsible for temporary memory storage, as in working memory, or that it may be involved in processing information regarding the timing of events. These models predict that abnormalities in SLP should lead to learning deficits. We tested this prediction in four lines of mutant mice with abnormal SLP, but apparently normal LTP-mice heterozygous for a alpha-calcium calmodulin kinase II mutation (alpha CaMKII +/-) have lower paired-pulse facilitation (PPF) and increased post-tetanic potentiation (PTP); mice lacking synapsin II (SyII-/-), and mice defective in both synapsin I and synapsin II (SyI/II-/-), show normal PPF but lower PTP; in contrast, mice just lacking synapsin I (SyI-/-) have increased PPF, but normal PTP. RESULTS: Our behavioral results demonstrate that alpha CaMKII +/-, SyII-/- and SyI/II-/- mutant mice, which have decreased PPF or PTP, have profound impairments in learning tasks. In contrast, behavioral analysis did not reveal learning deficits in SyI-/- mice, which have increased PPF. CONCLUSIONS: Our results are consistent with models that propose a role for SLP in learning, as mice with decreased PPF or PTP, in the absence of known LTP deficits, also show profound learning impairments. Importantly, analysis of the SyI-/- mutants demonstrated that an increase in PPF does not disrupt learning.

Cochlear and trigeminal systems contributing to the startle reflex in rats.

The startle reflex is evoked by strong acoustic or tactile stimuli, or by electrical stimulation of acoustic or tactile pathways. To dissociate the contributions of acoustic and tactile pathways, stimulating electrodes were placed in adjacent cochlear and trigeminal nuclei. The currents needed to evoke startle-like responses were an order of magnitude lower in ventral trigeminal sites (12-80 microA for a 0.1-ms pulse) than in cochlear nucleus sites (150-800 microA). At low threshold sites in both areas, brief acoustic stimuli were followed 0-4 ms later by a single electrical pulse and the current required to evoke startle was measured at several interstimulus intervals. Summation between the acoustic and electrical stimuli for startle was strong in both cochlear and trigeminal sites. Collision effects were found in the anteroventral cochlear nucleus when the electrical stimulus followed the ipsilateral acoustic stimulus by 2.0 ms, suggesting that acoustic startle is mediated by axons in the anteroventral cochlear nucleus. Collision effects were found at 4.0 ms if the electrical stimulus was presented in the contralateral pontine reticular formation, suggesting that acoustic signals mediating startle mainly cross to the pontine reticular formation. Collision effects were not found in medial or posterior sites in the cochlear nucleus, or trigeminal sites, suggesting that the neurons that mediate startle in these sites do not mediate acoustic startle. Therefore, acoustic startle is mediated through high threshold cochlear nucleus sites, while low threshold sites are non-acoustic, probably as a result of trigeminal or vestibular stimulation.

Circadian modulation in the rat acoustic startle circuit.

The acoustic startle reflex (ASR) in rats exhibits robust circadian modulation, with ASR amplitudes greater during subjective night. To identify the location of this modulation, startle reactions were evoked either acoustically or electrically via electrodes implanted in the primary ASR circuit. Startle amplitudes were compared at different times in the circadian cycle. In constant environmental conditions, startle amplitudes were greater in subjective night for acoustically evoked and for electrically evoked reactions from the ventral lateral lemniscus and medial longitudinal fasciculus. The results show that at least 1 site of circadian modulation must occur at some point in the circuit after the last brainstem synapse in the caudal pontine reticular formation, at the level of spinal interneurons or motoneurons or at the neuromuscular junction.

Fear-potentiated startle and electrically evoked startle mediated by synapses in rostrolateral midbrain.

Startle amplitudes are increased when acoustic startle responses are elicited in the presence of a stimulus that has previously been paired with shock. This "fear-potentiated" startle response appears to be mediated via the caudal ventral amygdalofugal pathway to the brainstem. Electrical stimulation of this pathway evokes unconditioned startlelike responses. Collision tests have shown that a monosynaptic connection from amygdala to midbrain mediates these responses. Collision tests here localize these synapses mediating electrically evoked startlelike responses to the rostrolateral midbrain in awake rats. To test whether rostrolateral midbrain synapses also mediate fear-potentiated startle, we lesioned cells in these sites with ibotenic acid. These lesions completely blocked fear potentiation of acoustic startle. These same lesions did not block potentiation of startle by d-amphetamine (6 mg/kg).

The dorsal hippocampus is essential for context discrimination but not for contextual conditioning.

The authors describe how (a) the timing of hippocampal lesions and (b) the behavioral-representational demands of the task affect the requirement for the hippocampus in contextual fear conditioning. Post- but not pretraining lesions of the hippocampus greatly reduced contextual fear conditioning. In contrast, pretraining lesions of the hippocampus abolished context discrimination, a procedure in which mice are trained to discriminate between 2 similar chambers (shock context vs. no-shock context). Whereas either contextual- or cue-based strategies can be used to recognize an aversive context, discrimination between similar contexts is optimally acquired by contextual (hippocampal)-based strategies. In keeping with the lesion results, Nf1(+/-)/Nmdar1(+/-) mutant mice, which have spatial learning deficits, are impaired in context discrimination but not in contextual conditioning. Together, these data dissociate hippocampal and nonhippocampal contributions to contextual conditioning, and they provide direct evidence that the hippocampus plays an essential role in the processing of contextual stimuli.

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.

Axons and synapses mediating electrically evoked startle: collision tests and latency analysis.

Davis et al. proposed that the primary acoustic startle reflex is mediated by synapses in the ventral cochlear nucleus (VCN), lateral lemniscus (LL) and caudal pontine reticular formation (PnC). The collision test was used here to estimate the locations of synapses mediating startle-like responses in these sites, and the conduction times between and across these synapses. Conditioning (C) and test (T) pulses were delivered to pairs of sites in chloral hydrate anaesthetized rats, and current thresholds for, and latencies of, hindlimb EMG responses were determined. When sites are axonally connected, thresholds increase at the same positive and negative C-T intervals ('symmetric collision'), but when sites are connected by strong synapses, thresholds increase at asymmetric C-T intervals. (i) Asymmetric collisions between VCN and contralateral PnC centered on a C-T interval of + 0.5 ms suggest a monosynaptic connection in ventrolateral pons (VLP), near LL. (ii) Asymmetric collisions between VCN and contralateral medulla centered on a C-T interval of + 0.85 to + 1.0 ms suggest a disynaptic connection. (iii) Asymmetric collisions between VLP and ipsilateral medulla centered on a C-T interval of 0.2-0.4 ms suggest a monosynaptic connection in PnC, as shown previously in freely behaving animals [32]. (4) Symmetric collisions between VLP and rostral PnC, and between caudal PnC and medulla suggest fast axonal connections. Latency differences between electrode pairs were generally similar to collision-derived conduction times. From these, conduction times and transmission times were estimated for each axon bundle and synapse in the circuit.

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.

Impact of early adverse experience on complexity of adult-generated neurons.

New neurons continue to be generated in the dentate gyrus (DG) region of the hippocampus throughout adulthood, and abnormal regulation of this process has emerged as an endophenotype common to several psychiatric disorders. Previous research shows that genetic risk factors associated with schizophrenia alter the maturation of adult-generated neurons. Here, we investigate whether early adversity, a potential environmental risk factor, similarly influences adult neurogenesis. During the first 2 weeks of postnatal life, mice were subject to repeated and unpredictable periods of separation from their mothers. When the mice reached adulthood, pharmacological and retroviral labelling techniques were used to assess the generation and maturation of new neurons. We found that adult mice that were repeatedly separated from their mothers early in life had similar rates of proliferation in the DG, but had fewer numbers of cells that survived and differentiated into neurons. Furthermore, neurons generated in adulthood had less complex dendritic arborization and fewer dendritic spines. These findings indicate that early adverse experience has a long-lasting impact on both the number and the complexity of adult-generated neurons in the hippocampus, suggesting that the abnormal regulation of adult neurogenesis associated with psychiatric disorders could arise from environmental influence alone, or from complex interactions of environmental factors with genetic predisposition.

The acoustic startle reflex: neurons and connections.

The startle reflex protects animals from blows or predatory attacks by quickly stiffening the limbs, body wall and dorsal neck in the brief time period before directed evasive or defensive action can be performed. The acoustic startle reflex in rats and cats is mediated primarily by a small cluster of giant neurons in the ventrocaudal part of the nucleus reticularis pontis caudalis (RPC) of the reticular formation. Activation of these RPC neurons occurs 3-8 ms after the acoustic stimulus reaches the ear. Undetermined neurons of the cochlear nuclei activate RPC via weak monosynaptic and strong disynaptic connections. The strong disynaptic input occurs via neurons of the contralateral ventrolateral pons, including large neurons of the ventrolateral tegmental nucleus that integrate auditory, tactile and vestibular information. RPC giant neurons, in turn, activate hundreds of motoneurons in the brain stem and the length of the spinal cord via large reticulospinal axons near the medial longitudinal fasciculus. To hindlimb motoneurons, monosynaptic connections from the reticulospinal tract are weak, but disynaptic connections via spinal cord interneurons are stronger and show temporal facilitation, like the startle response itself.

Long-term memory underlying hippocampus-dependent social recognition in mice.

The ability to learn and remember individuals is critical for the stability of social groups. Social recognition reflects the ability of mice to identify and remember conspecifics. Social recognition is assessed as a decrease in spontaneous investigation behaviors observed in a mouse reexposed to a familiar conspecific. Our results demonstrate that group-housed mice show social memory for a familiar juvenile when tested immediately, 30 min, 24 h, 3 days, and 7 days after a single 2-min-long interaction. Interestingly, chronic social isolation disrupts long-term, but not 30-min, social memory. Even a 24-h period of isolation disrupts long-term social memory, a result that may explain why previous investigators only observed short-term social memory in individually housed rodents. Although it has no obvious configural, relational, or spatial characteristics, here we show that social memory shares characteristics of other hippocampus-dependent memories. Ibotenic acid lesions of the hippocampus disrupt social recognition at 30 min, but not immediately after training. Furthermore, long-term, but not short-term social memory is dependent on protein synthesis and cyclic AMP responsive element binding protein (CREB) function. These results outline behavioral, systems, and molecular determinants of social recognition in mice, and they suggest that it is a powerful paradigm to investigate hippocampal learning and memory.

CREB and memory.

The cAMP responsive element binding protein (CREB) is a nuclear protein that modulates the transcription of genes with cAMP responsive elements in their promoters. Increases in the concentration of either calcium or cAMP can trigger the phosphorylation and activation of CREB. This transcription factor is a component of intracellular signaling events that regulate a wide range of biological functions, from spermatogenesis to circadian rhythms and memory. Here we review the key features of CREB-dependent transcription, as well as the involvement of CREB in memory formation. Evidence from Aplysia, Drosophila, mice, and rats shows that CREB-dependent transcription is required for the cellular events underlying long-term but not short-term memory. While the work in Aplysia and Drosophila only involved CREB function in very simple forms of conditioning, genetic and pharmacological studies in mice and rats demonstrate that CREB is required for a variety of complex forms of memory, including spatial and social learning, thus indicating that CREB may be a universal modulator of processes required for memory formation.

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.

Alpha-CaMKII-dependent plasticity in the cortex is required for permanent memory.

Cortical plasticity seems to be critical for the establishment of permanent memory traces. Little is known, however, about the molecular and cellular processes that support consolidation of memories in cortical networks. Here we show that mice heterozygous for a null mutation of alpha-calcium-calmodulin kinase II (alpha-CaMKII+/-) show normal learning and memory 1-3 days after training in two hippocampus-dependent tasks. However, their memory is severely impaired at longer retention delays (10-50 days). Consistent with this, we found that alpha-CaMKII+/- mice have impaired cortical, but not hippocampal, long-term potentiation. Our results represent a first step in unveiling the molecular and cellular mechanisms underlying the establishment of permanent memories, and they indicate that alpha-CaMKII may modulate the synaptic events required for the consolidation of memory traces in cortical networks.

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.

Molecular, cellular, and neuroanatomical substrates of place learning.

Learning and remembering the location of food resources, predators, escape routes, and immediate kin is perhaps the most essential form of higher cognitive processing in mammals. Two of the most frequently studied forms of place learning are spatial learning and contextual conditioning. Spatial learning refers to an animal's capacity to learn the location of a reward, such as the escape platform in a water maze, while contextual conditioning taps into an animal's ability to associate specific places with aversive stimuli, such as an electric shock. Recently, transgenic and gene targeting techniques have been introduced to the study of place learning. In contrast with the abundant literature on the neuroanatomical substrates of place learning in rats, very little has been done in mice. Thus, in the first part of this article, we will review our studies on the involvement of the hippocampus in both spatial learning and contextual conditioning. Having demonstrated the importance of the hippocampus to place learning, we will then focus attention on the molecular and cellular substrates of place learning. We will show that just as in rats, mouse hippocampal pyramidal cells can show place specific firing. Then, we will review our evidence that hippocampal-dependent place learning involves a number of interacting physiological mechanisms with distinct functions. We will show that in addition to long-term potentiation, the hippocampus uses a number of other mechanisms, such as short-term-plasticity and changes in spiking, to process, store, and recall information. Much of the focus of this article is on genetic studies of learning and memory (L&M). However, there is no single experiment that can unambiguously connect any cellular or molecular mechanism with L&M. Instead, several different types of studies are required to determine whether any one mechanism is involved in L&M, including (i) the development of biologically based learning models that explain the involvement of a given mechanism in L&M, (ii) lesion experiments (genetics and pharmacology), (iii) direct observations during learning, and (iv) experiments where learning is triggered by turning on the candidate mechanism. We will show how genetic techniques will be key to unraveling the molecular and cellular basis of place learning.

Sensorimotor gating abnormalities in young males with fragile X syndrome and Fmr1-knockout mice.

Fragile X syndrome (FXS) is the most common single gene (FMR1) disorder affecting cognitive and behavioral function in humans. This syndrome is characterized by a cluster of abnormalities including lower IQ, attention deficits, impairments in adaptive behavior and increased incidence of autism. Here, we show that young males with FXS have profound deficits in prepulse inhibition (PPI), a basic marker of sensorimotor gating that has been extensively studied in rodents. Importantly, the magnitude of the PPI impairments in the fragile X children predicted the severity of their IQ, attention, adaptive behavior and autistic phenotypes. Additionally, these measures were highly correlated with each other, suggesting that a shared mechanism underlies this complex phenotypic cluster. Studies in Fmr1-knockout mice also revealed sensorimotor gating and learning abnormalities. However, PPI and learning were enhanced rather than reduced in the mutants. Therefore, these data show that mutations of the FMR1 gene impact equivalent processes in both humans and mice. However, since these phenotypic changes are opposite in direction, they also suggest that murine compensatory mechanisms following loss of FMR1 function differ from those in humans.