Contributions of anterior cingulate cortex and basolateral amygdala to decision confidence and learning under uncertainty.

The subjective sense of certainty, or confidence, in ambiguous sensory cues can alter the interpretation of reward feedback and facilitate learning. We trained rats to report the orientation of ambiguous visual stimuli according to a spatial stimulus-response rule that must be learned. Following choice, rats could wait a self-timed delay for reward or initiate a new trial. Waiting times increase with discrimination accuracy, demonstrating that this measure can be used as a proxy for confidence. Chemogenetic silencing of BLA shortens waiting times overall whereas ACC inhibition renders waiting times insensitive to confidence-modulating attributes of visual stimuli, suggesting contribution of ACC but not BLA to confidence computations. Subsequent reversal learning is enhanced by confidence. Both ACC and BLA inhibition block this enhancement but via differential adjustments in learning strategies and consistent use of learned rules. Altogether, we demonstrate dissociable roles for ACC and BLA in transmitting confidence and learning under uncertainty.

Age-dependent modulation of hippocampal long-term potentiation by antioxidant enzymes.

Oxidative stress has long been associated with normal aging and age-related neurodegenerative disorders such as Alzheimer's disease (AD) and Parkinson's disease (PD). However, it is now evident that reactive oxygen species (ROS) such as superoxide (O(2-*)) and hydrogen peroxide (H(2)O(2)) also play pivotal roles in normal cell signaling. The focus of the present study was to examine the effects of the antioxidant enzymes CuZnSOD (SOD1) and catalase, which produce and remove H(2)O(2), respectively, on long-term potentiation (LTP) forms of synaptic plasticity during aging. Consistent wth previous studies, LTP, when induced in vitro in CA1 of the hippocampus with a high-frequency stimulation protocol, is significantly reduced in slices from older mice (22-26 months) relative to younger mice (2-4 months). Neither knockout of the endogenous catalase gene (Cat KO) nor acute enzymatic treatment with SOD1 altered LTP in slices from adult mice. Conversely, enzymatic applications of SOD1 inhibited LTP in slices from older mice. A much different set of results emerges with exogenous applications of catalase to hippocampal slices. Catalase significantly inhibited LTP in slices from adult mice but reversed age-related LTP deficits in slices from older mice. Measurements of H(2)O(2) showed that exogenous treatments with catalase lowered H(2)O(2) in synapse-enriched synaptoneurosome (SN) fractions prepared from adult mice. Notably, SNs from both Cat KO and old mice were deficient in removing extracellular challenges of H(2)O(2). Overall, the results suggest that dynamic alterations in extracellular H(2)O(2) metabolism affect synaptic plasticity in the hippocampus during aging.

A novel role for cyclic guanosine 3',5'monophosphate signaling in synaptic plasticity: a selective suppressor of protein kinase A-dependent forms of long-term potentiation.

At excitatory synapses onto hippocampal CA1 pyramidal cells, activation of cyclic AMP-dependent protein kinase and subsequent down-regulation of protein phosphatases has a crucial role in the induction of long-term potentiation by low-frequency patterns of synaptic stimulation. Because the second messenger cyclic guanosine 3',5'monophosphate can regulate the activity of different forms of the cyclic AMP degrading enzyme phosphodiesterase, we examined whether increases in cyclic guanosine 3',5'monophosphate can modulate long-term potentiation induction in the mouse hippocampal CA1 region through effects on cyclic AMP signaling. Using the cyclic guanosine 3',5'monophosphate-specific phosphodiesterase inhibitor zaprinast or the nitric oxide donor S-nitroso-D,L-penicillamine to elevate cyclic guanosine 3',5'monophosphate levels we found that increases in cyclic guanosine 3',5'monophosphate strongly inhibit the induction of long-term potentiation by low-frequency patterns of synaptic stimulation where protein kinase A activation is required for long-term potentiation induction. In contrast, zaprinast and S-nitroso-D,L-penicillamine had no effect on the induction of long-term potentiation by high-frequency patterns of synaptic stimulation that induce long-term potentiation in a protein kinase A-independent manner. Directly activating protein kinase A with the phosphodiesterase-resistant cyclic AMP analog 8-Br-cAMP, blocking all phosphodiesterases with 3-isobutyl-1-methylxanthine, or inhibiting protein phosphatases rescued long-term potentiation induction in zaprinast-treated slices. Together, these results suggest that increases in cyclic guanosine 3',5'monophosphate inhibit long-term potentiation by activating phosphodiesterases that interfere with the protein kinase A-mediated suppression of protein phosphatases needed for long-term potentiation induction. Consistent with the notion that this cyclic guanosine 3',5'monophosphate-mediated inhibitory pathway is recruited by some patterns of synaptic activity, blocking cyclic guanosine 3',5'monophosphate production strongly facilitated the induction of long-term potentiation by long trains of theta-frequency synaptic stimulation. Together, our results indicate that increases in cyclic guanosine 3',5'monophosphate can act as a long-term potentiation suppressor mechanism that selectively constrains the induction of protein kinase A-dependent forms of long-term potentiation induced by low-frequency patterns of synaptic stimulation.

Adenylyl cyclase-dependent form of chemical long-term potentiation triggers translational regulation at the elongation step.

The persistent maintenance of long-term potentiation requires both messenger RNA and protein synthesis. While there is mounting evidence for an active role of protein synthesis in hippocampal long-term potentiation, the nature of mechanisms underlying its regulation has not yet been established. We used a previously described chemical long-term potentiation protocol [J Neurosci 19 (1999) 2500] to address the hypothesis that signaling mechanisms, involved in long-lasting long-term potentiation, directly regulate protein synthesis. Chemical long-term potentiation is an N-methyl-D-aspartate receptor-dependent form of plasticity, which relies on both synaptic activity, in the form of spontaneous bursting induced by high concentrations of K(+) and Ca(2+), and cyclic AMP/adenylyl cyclase signaling. We found that chemical long-term potentiation in CA1 of the mouse hippocampus lasts for at least 3 hours and requires both messenger RNA and protein synthesis. However, surprisingly de novo total protein synthesis was paradoxically decreased at 1 hour after long-term potentiation induction. Consistent with the decrease in total protein synthesis in potentiated CA1, phosphorylation of eukaryotic elongation factor 2 was increased and is likely responsible for inhibition of translation at the elongation step. Increased phosphorylation of eukaryotic elongation factor 2 was dependent on coincident cyclic AMP/adenylyl cyclase activation and synaptic activity and required N-methyl-D-aspartate receptor activation. Despite the inhibition in total protein synthesis, the level of the immediate early gene protein Arc (activity regulated cytoskeleton-associated protein) increased at 1 hour after chemical long-term potentiation induction. Taken together, the results suggest that regulation at the elongation step of protein synthesis contributes to persistent forms of long-term potentiation.

Age-related deficits in long-term potentiation are insensitive to hydrogen peroxide: coincidence with enhanced autophosphorylation of Ca2+/calmodulin-dependent protein kinase II.

Reactive oxygen species (ROS) can have deleterious effects for both normal aging and Alzheimer's disease (AD). We examined the hypothesis that synapses undergoing long-term potentiation (LTP) are preferentially at risk for ROS-mediated oxidative stress during aging. We observed age-dependent deficits in LTP induced by a high-frequency stimulation (HFS) protocol in the CA1 region of hippocampus from C57BL/6 mice. There was a significant difference between LTP measured over 60 min in young (1-2 months) and old (23-26 months) mice. In oxidative stress studies, exogenous H(2)O(2) (580 micro M) significantly inhibited LTP in young mice; a similar dose of H(2)O(2) failed to inhibit LTP in slices from adult (2-4 months) or from old mice. The results show that there are significant deficits in LTP in aging mice, but such deficits are insensitive to H(2)O(2). Western immunoblotting studies in young mice show that the relative levels of autophosphorylated alpha-Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) are unchanged in hippocampal CA1 treated with H(2)O(2) relative to untreated controls. However with aging, there is a significant enhancement in the levels of autophosphorylated CaMKII in H(2)O(2)-treated CA1 of older mice. Phosphorylation of RC3/neurogranin (Ng) by protein kinase C (PKC) is decreased in CA1 in response to H(2)O(2) treatment, irrespective of age. We propose that, during aging, enhanced local release of H(2)O(2) from mitochondria may induce a compensatory "ceiling" effect at synapses, so that the levels of autophosphorylated alpha CaMKII are aberrantly saturated, leading to alterations in synaptic plasticity.

Multiprotein complex signaling and the plasticity problem.

Synaptic transmission of distinct patterns of spikes, or 'neural code', leads to plastic changes in synapses and other parts of the neuron, as well as learning in animals. Recent findings indicate that specialized multiprotein structures associated with neurotransmitter receptors and cell-adhesion proteins function as molecular devices that both read the neural code and initiate long-term changes in synaptic structure and function.

Coactivation of beta-adrenergic and cholinergic receptors enhances the induction of long-term potentiation and synergistically activates mitogen-activated protein kinase in the hippocampal CA1 region.

Interactions between noradrenergic and cholinergic receptor signaling may be important in some forms of learning. To investigate whether noradrenergic and cholinergic receptor interactions regulate forms of synaptic plasticity thought to be involved in memory formation, we examined the effects of concurrent beta-adrenergic and cholinergic receptor activation on the induction of long-term potentiation (LTP) in the hippocampal CA1 region. Low concentrations of the beta-adrenergic receptor agonist isoproterenol (ISO) and the cholinergic receptor agonist carbachol had no effect on the induction of LTP by a brief train of 5 Hz stimulation when applied individually but dramatically facilitated LTP induction when coapplied. Although carbachol did not enhance ISO-induced increases in cAMP, coapplication of ISO and carbachol synergistically activated p42 mitogen-activated protein kinase (p42 MAPK). This suggests that concurrent beta-adrenergic and cholinergic receptor activation enhances LTP induction by activating MAPK and not by additive or synergistic effects on adenylyl cyclase. Consistent with this, blocking MAPK activation with MEK inhibitors suppressed the facilitation of LTP induction produced by concurrent beta-adrenergic and cholinergic receptor activation. Although MEK inhibitors also suppressed the induction of LTP by a stronger 5 Hz stimulation protocol that induced LTP in the absence of ISO and carbachol, they had no effect on LTP induced by high-frequency synaptic stimulation or low-frequency synaptic stimulation paired with postsynaptic depolarization. Our results indicate that MAPK activation has an important, modulatory role in the induction of LTP and suggest that coactivation of noradrenergic and cholinergic receptors regulates LTP induction via convergent effects on MAPK.

Adenylyl cyclase activation modulates activity-dependent changes in synaptic strength and Ca2+/calmodulin-dependent kinase II autophosphorylation.

Activation of the Ca2+- and calmodulin-dependent protein kinase II (CaMKII) and its conversion into a persistently activated form by autophosphorylation are thought to be crucial events underlying the induction of long-term potentiation (LTP) by increases in postsynaptic Ca2+. Because increases in Ca2+ can also activate protein phosphatases that oppose persistent CaMKII activation, LTP induction may also require activation of signaling pathways that suppress protein phosphatase activation. Because the adenylyl cyclase (AC)-protein kinase A signaling pathway may provide a mechanism for suppressing protein phosphatase activation, we investigated the effects of AC activators on activity-dependent changes in synaptic strength and on levels of autophosphorylated alphaCaMKII (Thr286). In the CA1 region of hippocampal slices, briefly elevating extracellular Ca2+ induced an activity-dependent, transient potentiation of synaptic transmission that could be converted into a persistent potentiation by the addition of phosphatase inhibitors or AC activators. To examine activity-dependent changes in alphaCaMKII autophosphorylation, we replaced electrical presynaptic fiber stimulation with an increase in extracellular K+ to achieve a more global synaptic activation during perfusion of high Ca2+ solutions. In the presence of the AC activator forskolin or the protein phosphatase inhibitor calyculin A, this treatment induced a LTP-like synaptic potentiation and a persistent increase in autophosphorylated alphaCaMKII levels. In the absence of forskolin or calyculin A, it had no lasting effect on synaptic strength and induced a persistent decrease in autophosphorylated alphaCaMKII levels. Our results suggest that AC activation facilitates LTP induction by suppressing protein phosphatases and enabling a persistent increase in the levels of autophosphorylated CaMKII.

A nitric oxide-independent and beta-adrenergic receptor-sensitive form of metaplasticity limits theta-frequency stimulation-induced LTP in the hippocampal CA1 region.

The induction of long-term potentiation (LTP) and long-term depression (LTD) at excitatory synapses in the hippocampus can be strongly modulated by patterns of synaptic stimulation that otherwise have no direct effect on synaptic strength. Likewise, patterns of synaptic stimulation that induce LTP or LTD not only modify synaptic strength but can also induce lasting changes that regulate how synapses will respond to subsequent trains of stimulation. Collectively known as metaplasticity, these activity-dependent processes that regulate LTP and LTD induction allow the recent history of synaptic activity to influence the induction of activity-dependent changes in synaptic strength and may thus have an important role in information storage during memory formation. To explore the cellular and molecular mechanisms underlying metaplasticity, we investigated the role of metaplasticity in the induction of LTP by theta-frequency (5-Hz) synaptic stimulation in the hippocampal CA1 region. Our results show that brief trains of theta-frequency stimulation not only induce LTP but also activate a process that inhibits the induction of additional LTP at potentiated synapses. Unlike other forms of metaplasticity, the inhibition of LTP induction at potentiated synapses does not appear to arise from activity-dependent changes in NMDA receptor function, does not require nitric oxide signaling, and is strongly modulated by beta-adrenergic receptor activation. Together with previous findings, our results indicate that mechanistically distinct forms of metaplasticity regulate LTP induction and suggest that one way modulatory transmitters may act to regulate synaptic plasticity is by modulating metaplasticity.

Enhanced long-term potentiation and impaired learning in mice with mutant postsynaptic density-95 protein.

Specific patterns of neuronal firing induce changes in synaptic strength that may contribute to learning and memory. If the postsynaptic NMDA (N-methyl-D-aspartate) receptors are blocked, long-term potentiation (LTP) and long-term depression (LTD) of synaptic transmission and the learning of spatial information are prevented. The NMDA receptor can bind a protein known as postsynaptic density-95 (PSD-95), which may regulate the localization of and/or signalling by the receptor. In mutant mice lacking PSD-95, the frequency function of NMDA-dependent LTP and LTD is shifted to produce strikingly enhanced LTP at different frequencies of synaptic stimulation. In keeping with neural-network models that incorporate bidirectional learning rules, this frequency shift is accompanied by severely impaired spatial learning. Synaptic NMDA-receptor currents, subunit expression, localization and synaptic morphology are all unaffected in the mutant mice. PSD-95 thus appears to be important in coupling the NMDA receptor to pathways that control bidirectional synaptic plasticity and learning.

Postsynaptic complex spike bursting enables the induction of LTP by theta frequency synaptic stimulation.

Long-term potentiation (LTP), a persistent enhancement of synaptic transmission that may be involved in some forms of learning and memory, is induced at excitatory synapses in the CA1 region of the hippocampus by coincident presynaptic and postsynaptic activity. Although action potentials back-propagating into dendrites of hippocampal pyramidal cells provide sufficient postsynaptic activity to induce LTP under some in vitro conditions, it is not known whether LTP can be induced by patterns of postsynaptic action potential firing that occur in these cells in vivo. Here we report that a characteristic in vivo pattern of action potential generation in CA1 pyramidal cells known as the complex spike burst enables the induction of LTP during theta frequency synaptic stimulation in the CA1 region of hippocampal slices maintained in vitro. Our results suggest that complex spike bursting may have an important role in synaptic processes involved in learning and memory formation, perhaps by producing a highly sensitive postsynaptic state during which even low frequencies of presynaptic activity can induce LTP.

5-Hz stimulation of CA3 pyramidal cell axons induces a beta-adrenergic modulated potentiation at synapses on CA1, but not CA3, pyramidal cells.

In mouse hippocampal slices, long-term potentiation (LTP) at Schaffer collateral fiber synapses onto CA1 pyramidal cells could be induced by brief trains of 5-Hz synaptic stimulation (30 s) or by longer trains of 5-Hz stimulation (3 min) delivered during beta-adrenergic receptor activation. In contrast, 5-Hz stimulation, either alone or in the presence of the beta-adrenergic receptor agonist isoproterenol, failed to induce LTP at associational-commissural (assoc-com) fiber synapses onto CA3 pyramidal cells. Our results suggest that although CA3 pyramidal cells give rise to both the Schaffer collateral fiber synapses in CA1 and the assoc-com fiber synapses in CA3, the induction of LTP at these synapses may be regulated by different activity- and modulatory neurotransmitter-dependent processes.

Functional screening of 2 Mb of human chromosome 21q22.2 in transgenic mice implicates minibrain in learning defects associated with Down syndrome.

Using Down syndrome as a model for complex trait analysis, we sought to identify loci from chromosome 21q22.2 which, when present in an extra dose, contribute to learning abnormalities. We generated low-copy-number transgenic mice, containing four different yeast artificial chromosomes (YACs) that together cover approximately 2 megabases (Mb) of contiguous DNA from 21q22.2. We subjected independent lines derived from each of these YAC transgenes to a series of behavioural and learning assays. Two of the four YACs caused defects in learning and memory in the transgenic animals, while the other two YACs had no effect. The most severe defects were caused by a 570-kb YAC; the interval responsible for these defects was narrowed to a 180-kb critical region as a consequence of YAC fragmentation. This region contains the human homologue of a Drosophila gene, minibrain, and strongly implicates it in learning defects associated with Down syndrome.

Activity-dependent beta-adrenergic modulation of low frequency stimulation induced LTP in the hippocampal CA1 region.

beta-Adrenergic receptor activation has a central role in the enhancement of memory formation that occurs during heightened states of emotional arousal. Although beta-adrenergic receptor activation may enhance memory formation by modulating long-term potentiation (LTP), a candidate synaptic mechanism involved in memory formation, the cellular basis of this modulation is not fully understood. Here, we report that, in the CA1 region of the hippocampus, beta-adrenergic receptor activation selectively enables the induction of LTP during long trains of 5 Hz synaptic stimulation. Protein phosphatase inhibitors mimic the effects of beta-adrenergic receptor activation on 5 Hz stimulation-induced LTP, suggesting that activation of noradrenergic systems during emotional arousal may enhance memory formation by inhibiting protein phosphatases that normally oppose the induction of LTP.

Hippocampal long-term potentiation is normal in heme oxygenase-2 mutant mice.

We have generated mice deficient in HO-2, the major cerebral isoform of heme oxygenase, in order to assess the potential role of carbon monoxide as a retrograde messenger in hippocampal LTP. Cerebral HO catalytic activity was markedly reduced in the HO-2 mutant mice, yet no differences were found between wild types and mutants in gross neuroanatomical structure, in basal hippocampal synaptic transmission, or in the amount of potentiation produced by various LTP induction protocols. Furthermore, zinc protoporphyrin IX, an inhibitor of HO, had nearly identical inhibitory effects on LTP in wild-type and HO-2 mutant hippocampal slices. Our data indicate that carbon monoxide produced endogenously by HO is unlikely to be a neuromodulator required for LTP in the hippocampus.

The molecular switch hypothesis fails to explain the inconsistent effects of the metabotropic glutamate receptor antagonist MCPG on long-term potentiation.

In the CA1 region of the hippocampus, the induction of long-term potentiation (LTP) appears to be controlled by a switch-like biochemical process that is persistently activated following metabotropic glutamate receptor (mGLUR) activation. However, the mGLUR antagonist (RS)-alpha-methyl-4-carboxyphenylglycine (MCPG) does not consistently block the induction of LTP, perhaps because the experimental conditions used by some investigators inadvertently activate this 'molecular switch', thereby fulfilling the requirement for mGLUR activation and rendering LTP insensitive to the effects of mGLUR antagonists. In mouse hippocampal slices we observed that MCPG does not block LTP induced by high-frequency stimulation, Moreover, stimulation protocols designed to deactivate an inadvertently activated molecular switch had no effect on the inability of MCPG to block LTP. MCPG (through a switch-independent mechanism) did inhibit the induction of LTP by a weak induction protocol. Our results thus suggest that MCPT-sensitive mGLURs are not required for the induction of LTP and that a mLGUR-activated 'molecular switch' does not explain the inconsistent effects of MCPG on LTP. Instead, MCPG-sensitive mGLURs may have a modulatory role in the induction of LTP that is most evident when LTP is induced by near threshold patterns of synaptic stimulation.

CaMKII regulates the frequency-response function of hippocampal synapses for the production of both LTD and LTP.

To investigate the function of the autophosphorylated form of CaMKII in synaptic plasticity, we generated transgenic mice that express a kinase that is Ca2+ independent as a result of a point mutation of Thr-286 to aspartate, which mimics autophosphorylation. Mice expressing the mutant form of the kinase show an increased level of Ca(2+)-independent CaMKII activity similar to that seen following LTP. The mice nevertheless exhibit normal LTP in response to stimulation at 100 Hz. However, at lower frequencies, in the range of 1-10 Hz, there is a systematic shift in the size and direction of the resulting synaptic change in the transgenic animals that favors LTD. The regulation of this frequency-response function by Ca(2+)-independent CaMKII activity seems to account for two previously unexplained synaptic phenomena, the relative loss of LTD in adult animals compared with juveniles and the enhanced capability for depression of facilitated synapses.

Endothelial NOS and the blockade of LTP by NOS inhibitors in mice lacking neuronal NOS.

Long-term potentiation (LTP) is a persistent increase in synaptic strength implicated in certain forms of learning and memory. In the CA1 region of the hippocampus, LTP is thought to involve the release of one or more retrograde messengers from the postsynaptic cell that act on the presynaptic terminal to enhance transmitter release. One candidate retrograde messenger is the membrane-permeant gas nitric oxide (NO), which in the brain is released after activation of the neuronal-specific NO synthase isoform (nNOS). To assess the importance of NO in hippocampal synaptic plasticity, LTP was examined in mice where the gene encoding nNOS was disrupted by gene targeting. In nNOS- mice, LTP induced by weak intensity tetanic stimulation was normal except for a slight reduction in comparison to that in wild-type mice and was blocked by NOS inhibitors, just as it was in wild-type mice. Immunocytochemical studies indicate that in the nNOS- mice as in wild-type mice, the endothelial form of NOS (eNOS) is expressed in CA1 neurons. These findings suggest that eNOS, rather than nNOS, generates NO within the postsynaptic cell during LTP.

Low-frequency stimulation erases LTP through an NMDA receptor-mediated activation of protein phosphatases.

In the CA1 region of adult guinea pig hippocampal slices, long trains of theta frequency (5 Hz) stimulation produced a small enhancement of basal synaptic transmission but depressed the strength of synaptic transmission at synapses that had recently undergone long-term potentiation (LTP). Five hertz stimulation delivered immediately prior to high-frequency stimulation also inhibited the subsequent induction of LTP. The depression of potentiated synapses by 5 Hz stimulation (depotentiation) was blocked by 2-amino-5-phosphonovalerate and was observed only during the early phases of LTP. Furthermore, the protein phosphatase inhibitors okadaic acid and calyculin A blocked both depotentiation and the ability of 5 Hz stimulation to inhibit subsequent LTP, suggesting that protein phosphatases are involved in the ability of 5 Hz stimulation to modulate synaptic plasticity in the CA1 region of the hippocampus.