Biomedical subjects
Publications and source records attributed to Richard F Thompson.
Long-term potentiation (LTP) and long-term depression (LTD) are arguably two of the most widely discussed cellular plasticity mechanisms for learning and memory. However, the extent to which they are required for behavioral plasticity and learning is not clear. In this issue of Neuron, Boyden et al. use mice lacking CaMKIV and Hansel et al. use mice lacking alphaCaMKII to assess the contribution of LTD to cerebellar learning.
The anterior interpositus nucleus (AIN) is the proposed site of memory formation of eyeblink conditioning. A large part of the underlying molecular events, however, remain unknown. To elucidate the molecular mechanisms, we examined transcriptional changes in the AIN of mice trained with delay eyeblink conditioning using microarray, quantitative real-time RT-PCR, and in situ hybridization techniques. Microarray analyses suggested that transcriptionally up-regulated gene sets were largely different between early (3-d training) and late (7-d) stages. Quantitative real-time RT-PCR aided by laser microdissection indicated that the expression of representative EARLY genes (Sgk, IkBa, and Plekhf1) peaked at 1-d training in both the paired and unpaired conditioning groups, and was maintained at a higher level in the paired group than in the unpaired group after 3-d training. In situ hybridization revealed increased expression of these genes in broad cerebellar areas, including the AIN, with no hemispheric preferences. In contrast, the expression of representative LATE genes (Vamp1, Camk2d, and Prkcd) was selectively increased in the AIN of the 7-d paired group, with dominance in the ipsilateral AIN. Increased Vamp1 mRNA expression was restricted to the ipsilateral dorsolateral hump, a subregion of the AIN. These expression patterns of two distinct subsets of genes fit well with the two-stage learning theory, which proposes emotional and motor learning phases, and support the notion that AIN has a crucial role in memory formation of eyeblink conditioning.
The conditioning context arises from the relatively static features of the training environment. In rabbit eyeblink conditioning, procedures that retard acquisition (conditioned stimulus [CS] preexposure, unconditioned stimulus preexposure, blocking manipulations) are attenuated by context changes. In this article the authors investigate the effect of context exposure after initial delay conditioning. After conditioned responses (CRs) were established, one group received 6 sessions of context exposure, whereas control groups either remained in their home cages or received exposure to handling and a novel context. Thereafter, all groups received CS-alone testing. The expression of CRs was substantially reduced following context exposure relative to any retention loss in the home-cage control. Exposure to handling and a novel context facilitated the CRs rather than reducing them.
Distinct neural regions may be engaged during acquisition and maintenance of some memories. In delay classical conditioning of the eyeblink response, the cerebellum is necessary for acquisition and expression of the conditioned response (CR), but loci of long-term memory storage are not known. Rabbits (Oryctolagus cuniculus) were trained, overtrained, and given either 30 additional days of training or 30 days of rest. Half the subjects in the rest group were given a reminder training session. Subjects then received either reversible inactivation of the cerebellar interpositus nucleus (muscimol) or permanent electrolytic lesions. In all cases, inactivation and lesions of the interpositus completely abolished the CR. The site of memory formation in the interpositus nucleus also appears to be the site of long-term memory storage.
The key issue in analyzing brain substrates of memory is the nature of memory traces, how memories are formed, stored, and retrieved in the brain. In order to analyze mechanisms of memory formation it is first necessary to find the loci of memory storage, the classic problem of localization. Various approaches to this issue are reviewed. A particular strategy is proposed that involves a number of different techniques (electrophysiological recording, lesions, electrical stimulation, pathway tracing) to identify the essential memory trace circuit for a given form of learning and memory. The methods of reversible inactivation can be used to localize the memory traces within this circuit. Using classical conditioning of eye blink and other discrete responses as a model system, the essential memory trace circuit is identified, the basic memory trace is localized (to the cerebellum), and putative higher-order memory traces are characterized in the hippocampus.
In this study, brain-derived neurotrophic factor (BDNF) heterozygous knock-outs were tested on fear conditioning, and their wild-type littermates were used as controls. Results showed that BDNF(+/-) mice are impaired in contextual learning, whereas tone learning remains intact. Because BDNF is involved in synaptic transmission and contextual learning is hippocampal dependent, we hypothesized that this deficit is attributable to abnormal BDNF-modulated synaptic plasticity in the hippocampus. A "gain-of-function" experiment was performed next by infusing recombinant BDNF protein into the hippocampal formation to investigate whether this deficit can be rescued. Infusion of BDNF protein into the hippocampus appeared to partially restore contextual fear learning of BDNF(+/-) mice. In conclusion, the present study suggests that BDNF plays a critical role in fear conditioning. Loss of one copy of the BDNF gene leads to impairment of contextual fear learning in BDNF(+/-). This deficit can be partially rescued by infusing BDNF protein into the hippocampus. Other brain regions interacting with the hippocampus in the context conditioned stimulus pathway, for example, the amygdala, may also require normal BDNF expression levels to fully rescue this impairment.
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Fos, the protein product of the immediate early gene c-fos, was used to map functional circuitry underlying contextual conditioned fear. Male rats were given footshocks in a distinctive context and later tested using freezing as the behavioral measure and compared with no-shock and no-retention-test control groups. An increased number of Fos-immunoreactive neurons was found in the lateral part of the central nucleus and in the anterior basolateral and lateral amygdalar nuclei in the brains of the conditioned-fear group compared with controls. Further, a greater number of Fos-immunoreactive neurons was observed in the right central and anterior basolateral nuclei compared with the number of labeled neurons in these structures on the left.
Rabbits (Oryctolagus cuniculus) were presented with 7 daily sessions of tone-alone training after conditioning. Before the beginning of each of the first 4 extinction sessions, an artificial tear solution or tetracaine hydrochloride was administered to the cornea of rabbits in the control group (n = 6) and experimental group (n = 7), respectively. There were no between-group differences in the percentage of conditioned responses between both groups. However, the amplitude of the conditioned response was notably reduced in the tetracaine group (M = 0.40, SEM +/- 0.216) relative to the control group (M = 1.32, SEM +/- 0.639) early in extinction. Results seem to suggest that although motor output has been found to play an important role in extinction, corneal sensory feedback is not necessary.
In contrast to our increasingly detailed understanding of how synaptic plasticity provides a cellular substrate for learning and memory, it is less clear how a neuron's voltage-gated ion channels interact with plastic changes in synaptic strength to influence behavior. We find, using generalized and regional knockout mice, that deletion of the HCN1 channel causes profound motor learning and memory deficits in swimming and rotarod tasks. In cerebellar Purkinje cells, which are a key component of the cerebellar circuit for learning of correctly timed movements, HCN1 mediates an inward current that stabilizes the integrative properties of Purkinje cells and ensures that their input-output function is independent of the previous history of their activity. We suggest that this nonsynaptic integrative function of HCN1 is required for accurate decoding of input patterns and thereby enables synaptic plasticity to appropriately influence the performance of motor activity.
The underlying neuronal substrates and behavioral properties that might mediate extinction of the classically conditioned eye-blink response (CR) were examined. Four groups of rabbits were trained to perform the CR. Two of the groups then received either three or six sessions of tone-alone extinction training while the motor nuclei that mediate expression of the CR (facial nucleus and accessory abducens) were reversibly inactivated with microinjections of the GABA agonist muscimol. After these inactivation extinction sessions, rabbits received four more extinction sessions without inactivation. Two groups of controls received either three or six extinction sessions while saline vehicle was infused into the motor nuclei, followed by four sessions with no infusions. Saline infusions had no effect on extinction, and controls extinguished the CR normally over the first three to four sessions. In contrast, muscimol inactivation of the motor nuclei completely prevented any performance of CRs during the three or six inactivation extinction sessions. At the start of the four extinction sessions without inactivation, rabbits performed CRs at the same rate and amplitude as controls on their first extinction sessions. The muscimol rabbits then extinguished the CR normally over the four sessions without inactivation. In short, inactivation of the motor nuclei completely prevented any extinction of the eye-blink CR with no effect on subsequent extinction without inactivation. These results are discussed in terms of possible neuroanatomical loci that might mediate the extinction process as well as how effects of manipulating CR performance during extinction may affect the extinction process.
Oxidative stress has been implicated in cognitive impairment in both old experimental animals and aged humans. This implication has led to the notion that antioxidant defense mechanisms in the brain are not sufficient to prevent age-related increase in oxidative damage and that dietary intake of a variety of antioxidants might be beneficial for preserving brain function. Here we report a dramatic loss of learning and memory function from 8 to 11 months of age in mice, associated with marked increases in several markers of brain oxidative stress. Chronic systemic administration of two synthetic catalytic scavengers of reactive oxygen species, Eukarion experimental compounds EUK-189 and EUK-207, from 8 to 11 months almost completely reversed cognitive deficits and increase in oxidative stress taking place during this time period in brain. In particular, increase in protein oxidation was completely prevented, whereas increase in lipid peroxidation was decreased by approximately 50%. In addition, we observed a significant negative correlation between contextual fear learning and levels of protein oxidation in brain. These results further support the role of reactive oxygen species in age-related learning impairment and suggest potential clinical applications for synthetic catalytic scavengers of reactive oxygen species.
17-beta-Estradiol (E2), by activating Src and ERK/MAP kinases, enhances NMDA receptor phosphorylation and function. NR2 subunits of NMDA receptors are truncated by calpain, an effect prevented by tyrosine phosphorylation of the subunits. The present study investigated whether E2-mediated activation of ERK and NR2 subunits phosphorylation were altered in 24-month-old female rats. Ovariectomy reduced ERK2 phosphorylation in brains from 3- but not 24-month-old female rats. In ovariectomized rats, restoration of estrogen levels increased ERK2 and NR2 phosphorylation in young but not aged animals. Calcium treatment of frozen-thawed brain sections decreased NR2 levels in both young and aged female rats. This effect was absent in E2-treated young ovariectomized female rats, but was not modified in aged ovariectomized female rats. These results indicate that E2 activation of ERK2 and NR2 phosphorylation is markedly reduced in aged female rats, whereas calpain-mediated truncation of NR2 subunits is not different in young and aged rats. They suggest that several key elements of the mechanisms involved in estrogen-mediated regulation of synaptic plasticity are altered in aged animals.
A significant number of brain neurons in the rabbit brain were immunostained with anti-rabbit gamma-immunoglobulin (IgG). IgG-positive neurons were often found in the cerebellum, lower brainstem and motor nuclei. Similar IgG-positive neurons were occasionally found in the hippocampus, cerebral cortex and midbrain, but not in the striatum and thalamus. These neurons showed very clear Golgi-like staining of soma and dendrites but IgG staining was absent from the cell nuclei and axons. In particular, groups of Purkinje neurons in the rabbit cerebellum showed strong IgG-positive staining. To confirm whether the staining reflected the existence of IgG molecules in these neurons, staining specificity was carefully evaluated. Staining was specifically eliminated by pre-absorption of the antibodies with the purified rabbit IgG. An antibody to the neural cell adhesion molecule (NCAM or CD56), a member of the immunoglobulin superfamily, exhibited a completely different pattern of staining as that for IgG. To determine whether IgG-like immunoreactivity was a general feature of mammalian brain, brain sections of rabbits, rats, and mice were immunostained with antibodies to IgGs of each of the three species. Similar IgG-positive neurons were observed in all three species, although the distribution and frequency was characteristic for each species. In rabbit brain, anti-rabbit IgG stained-neurons were more abundant compared to rat and mouse brain. IgG-positive microglia-like cells were evident in mouse brain, but less frequent in rabbit and were hardly observed in rat brain. To evaluate whether stained neurons could synthesize IgG, in situ hybridization was carried out using an antisense oligonucleotide probe to rabbit IgG DNA. No significant label was observed in cerebellum. These results suggest that a significant number of neurons in the intact rabbit brain take up IgGs and concentrate them in their cytoplasm, although the molecular uptake mechanism is retained for future studies. Our results also suggest that the rabbit may be a suitable animal to study the function(s) of IgG in brain neurons.
The cerebellum is considered a brain structure in which memories for learned motor responses (e.g., conditioned eyeblink responses) are stored. Within the cerebellum, however, the relative importance of the cortex and the deep nuclei in motor learning/memory is not entirely clear. In this study, we show that the cerebellar cortex exerts both basal and stimulus-activated inhibition to the deep nuclei. Sequential application of a gamma-aminobutyric acid type A receptor (GABA(A)R) agonist and a noncompetitive GABA(A)R antagonist allows selective blockade of stimulus-activated inhibition. By using the same sequential agonist and antagonist methods in behaving animals, we demonstrate that the conditioned response (CR) expression and timing are completely dissociable and involve different inhibitory inputs; although the basal inhibition modulates CR expression, the conditioned stimulus-activated inhibition is required for the proper timing of the CR. In addition, complete blockade of cerebellar deep nuclear GABA(A)Rs prevents CR acquisition. Together, these results suggest that different aspects of the memories for eyeblink CRs are encoded in the cerebellar cortex and the cerebellar deep nuclei.
The interrelationships of cerebellar and cerebral neural circuits in the eyeblink paradigm were explored with the controlled application of cortical spreading depression (CSD) and lidocaine in the New Zealand albino rabbit. The initial research focus was directed toward the involvement of the motor cortex in the conditioned eyeblink response. However, CSD timing and triangulation results indicate that other areas in the cerebral cortex, particularly the auditory cortex (acoustic conditioned stimulus), appear to be critical for the CSD effect on the eyeblink response. In summary: (1) CSD can be elicited, monitored, and timed and its side effects controlled in 97% of awake rabbits in the right and/or left cerebral hemisphere(s) during eyeblink conditioning. (2) The motor cortex appears to play little or no part in classical conditioning of the eyeblink in the rabbit in the delay paradigm. (3) Inactivating the auditory cortex with CSD or lidocaine temporarily impairs the conditioned response during the first 5 to 15 days of training, but has little effect past that point.
A large body of evidence indicates that the cerebellum is essential for the acquisition, retention, and expression of the standard delay conditioned eyeblink response and that the basic memory trace appears to be established in the anterior interpositus nucleus (IP). Adaptive timing of the conditioned response (CR) is a prominent feature of classical conditioning-the CR peaks at the time of onset of the unconditioned stimulus (US) over a wide range of CS-US interstimulus intervals (ISI). A key issue is whether this timing is established by the cerebellar circuitry or prior to the cerebellum. In this study timing of conditioned eyeblink responses established via electrical stimulation of the interpositus nucleus as a conditioned stimulus (CS) was analyzed prior to and following modification of the CS-US interval in well-trained rabbits. Consistent with previous results, learning under these conditions is very rapid and robust. The CR peak eyeblink latencies are initially timed to the US onset and adjust accordingly to lengthening or shortening of the CS-US interval, just as with peripheral CSs. The acquisition of conditioned eyeblink responses by direct electrical stimulation of the IP as a CS thus retains temporal flexibility following shifts in the CS-US delay, as found in standard classical eyeblink conditioning procedures.
Classical conditioning of the eyeblink reflex to a neutral stimulus that predicts an aversive stimulus is a basic form of associative learning. Acquisition and retention of this learned response require the cerebellum and associated sensory and motor pathways and engage several other brain regions including the hippocampus, neocortex, neostriatum, septum, and amygdala. The cerebellum and its associated circuitry form the essential neural system for delay eyeblink conditioning. Trace eyeblink conditioning, a learning paradigm in which the conditioned and unconditioned stimuli are noncontiguous, requires both the cerebellum and the hippocampus and exhibits striking parallels to declarative memory formation in humans. Identification of the neural structures critical to the development and maintenance of the conditioned eyeblink response is an essential precursor to the investigation of the mechanisms responsible for the formation of these associative memories. In this review, we describe the evidence used to identify the neural substrates of classical eyeblink conditioning and potential mechanisms of memory formation in critical regions of the hippocampus and cerebellum. Addressing a central goal of behavioral neuroscience, exploitation of this simple yet robust model of learning and memory has yielded one of the most comprehensive descriptions to date of the physical basis of a learned behavior in mammals.