Conditioning-induced Changes Research Articles

Formation of memory assemblies through the DNA-sensing TLR9 pathway

As hippocampal neurons respond to diverse types of information1, a subset assembles into microcircuits representing a memory2. Those neurons typically undergo energy-intensive molecular adaptations, occasionally resulting in transient DNA damage3–5. Here we found discrete clusters of excitatory hippocampal CA1 neurons with persistent double-stranded DNA (dsDNA) breaks, nuclear envelope ruptures and perinuclear release of histone and dsDNA fragments hours after learning. Following these early events, some neurons acquired an inflammatory phenotype involving activation of TLR9 signalling and accumulation of centrosomal DNA damage repair complexes6. Neuron-specific knockdown of Tlr9 impaired memory while blunting contextual fear conditioning-induced changes of gene expression in specific clusters of excitatory CA1 neurons. Notably, TLR9 had an essential role in centrosome function, including DNA damage repair, ciliogenesis and build-up of perineuronal nets. We demonstrate a novel cascade of learning-induced molecular events in discrete neuronal clusters undergoing dsDNA damage and TLR9-mediated repair, resulting in their recruitment to memory circuits. With compromised TLR9 function, this fundamental memory mechanism becomes a gateway to genomic instability and cognitive impairments implicated in accelerated senescence, psychiatric disorders and neurodegenerative disorders. Maintaining the integrity of TLR9 inflammatory signalling thus emerges as a promising preventive strategy for neurocognitive deficits.

Operant Training for Highly Palatable Food Alters Translating Messenger RNA in Nucleus Accumbens D2 Neurons and Reveals a Modulatory Role of Ncdn

BackgroundHighly palatable food triggers behavioral responses including strong motivation. These effects involve the reward system and dopamine neurons, which modulate neurons in the nucleus accumbens (NAc). The molecular mechanisms underlying the long-lasting effects of highly palatable food on feeding behavior are poorly understood. MethodsWe studied the effects of 2-week operant conditioning of mice with standard or isocaloric highly palatable food. We investigated the behavioral responses and dendritic spine modifications in the NAc. We compared the translating messenger RNA in NAc neurons identified by the type of dopamine receptors they express, depending on the kind of food and training. We tested the consequences of invalidation of an abundant downregulated gene, Ncdn. ResultsOperant conditioning for highly palatable food increased motivation for food even in well-fed mice. In wild-type mice, free choice between regular and highly palatable food increased weight compared with access to regular food only. Highly palatable food increased spine density in the NAc. In animals trained for highly palatable food, translating messenger RNAs were modified in NAc neurons expressing dopamine D2 receptors, mostly corresponding to striatal projection neurons, but not in neurons expressing D1 receptors. Knockout of Ncdn, an abundant downregulated gene, opposed the conditioning-induced changes in satiety-sensitive feeding behavior and apparent motivation for highly palatable food, suggesting that downregulation may be a compensatory mechanism. ConclusionsOur results emphasize the importance of messenger RNA alterations in D2 striatal projection neurons in the NAc in the behavioral consequences of highly palatable food conditioning and suggest a modulatory contribution of Ncdn downregulation.

Conditioning‑induced changes in sensory cortical maps detected in mice by intrinsic signal optical imaging.

Intrinsic signal optical imaging (ISOI) has been used previously for the detection of changes in sensory processing in the somatosensory cortex in response to environment alteration or after deprivation of sensory information. To date, there have been no reports of ISOI being used in learning‑induced changes in the somatosensory cortex. In the present study, ISOI was performed twice in the same mouse: before and after conditional fear learning. The conditioning paradigm consisted of pairing sensory stimulation of vibrissae with electric tail shock. In order to map the cortical representation of the vibrissa B1 with ISOI, we deflected the vibrissa with an intensive stimulation (frequency of 10 Hz for 6 s). After conditioning, we found that the cortical representation of vibrissa B1 had expanded by an average of 44%, compared with pre‑learning, by using images obtained with ISOI. Previously, we demonstrated an enlargement of the cortical representation of the vibrissae stimulated by the same behavioral training paradigm but using [14C]2‑deoxyglucose. This current investigation provides the first ISOI‑based evidence of learning‑induced changes in plasticity in the barrel cortex. The results indicate that irrespective of physiological mechanisms used for visualization of the vibrissae representation or subject's testing state (aware or anesthetized animal), the conditioning induced changes in each case in the cortical processing of intensive stimuli. This suggests specific functional reorganization of the neuronal circuits. Moreover, ISOI as a noninvasive method of mapping cortical activation in the same animal before and after behavioral training could serve as a very useful tool for precise manipulation within the cortex and for assessing the resulting effects on experience‑dependent cortical plasticity.

Disruptions in network plasticity precede deficits in memory following inhibition of retinoid signaling.

Retinoic acid, the active metabolite of vitamin A, is important for vertebrate cognition and hippocampal plasticity, but few studies have examined its role in invertebrate learning and memory, and its actions in the invertebrate central nervous system are currently unknown. Using the mollusc Lymnaea stagnalis, we examined operant conditioning of the respiratory behavior, controlled by a well-defined central pattern generator (CPG), and used citral to inhibit retinoic acid signaling. Both citral- and vehicle-treated animals showed normal learning, but citral-treated animals failed to exhibit long-term memory at 24 h. Cohorts of citral- or vehicle-treated animals were dissected into semi-intact preparations, either 1 h after training, or after the memory test 24 h later. Simultaneous electrophysiological recordings from the CPG pacemaker cell (right pedal dorsal 1; RPeD1) and an identified motorneuron (VI) were made while monitoring respiratory activity (pneumostome opening). Activity of the CPG pneumostome opener interneuron (input 3 interneuron; IP3) was also monitored indirectly. Vehicle-treated conditioned preparations showed significant changes in network parameters immediately after learning, such as reduced motorneuron bursting activity (from IP3 input), delayed pneumostome opening, and decoupling of coincident IP3 input within the network. However, citral-treated preparations failed to exhibit these network changes and more closely resembled naïve preparations. Importantly, these citral-induced differences were manifested immediately after training and before any overt changes in the behavioral response (memory impairment). These studies shed light on where and when retinoid signaling might affect a central pattern-generating network to promote memory formation during conditioning of a homeostatic behavior.NEW & NOTEWORTHY We provide novel evidence for how conditioning-induced changes in a CPG network are disrupted when retinoid signaling is inhibited. Inhibition of retinoic acid signaling prevents long-term memory formation following operant conditioning, but has no effect on learning. Simultaneous electrophysiological and behavioral analyses indicate network changes immediately following learning, but these changes are prevented with inhibition of retinoid signaling, before any overt changes in behavior. These data suggest sites for retinoid actions during memory formation.

Short-term sleep deprivation immediately after contextual conditioning inhibits BDNF signaling and disrupts memory consolidation in predator odor trauma mice model of PTSD

Post-traumatic stress disorder (PTSD) is a debilitating neuropsychiatric illness affecting > 7 million people every year in the US. Recently, we have shown that the mouse model of predator odor trauma (POT) displayed contextual conditioning and core features of PTSD including sleep disturbances (hyperarousal) and retrieval of traumatic memories following exposure to objective reminders (re-experiencing). PTSD is a disorder of memory function. Since memory consolidation requires the expression of BDNF along with an activation of MAPK/pERK signaling pathway in limbic brain structures (hippocampus and amygdala) and sleep favors memory consolidation, we hypothesized that short-term sleep deprivation (SD, 3 h), immediately after contextual conditioning will attenuate molecular correlates of memory consolidation, sleep disturbances, and memory consolidation. We performed two experiments in adult male C57BL/6J mice to test our hypothesis. Experiment 1 determined the effects of SD on contextual conditioning and changes in sleep wakefulness. Experiment 2 determined the effects of SD on contextual conditioning-induced changes in the expression of BDNF and pERK in hippocampus and amygdala. SD immediately after contextual conditioning (POT + SD group) significantly attenuated sleep disturbances, memory retrieval, and expression of pERK and BDNF in the hippocampus and amygdala as compared to POT-SD group (no SD after contextual conditioning). No significant differences were observed between POT + SD, NOC-SD (no contextual conditioning + no SD), and NOC + SD (no contextual conditioning + SD) groups. Memory consolidation requires sleep and the expression of pERK and BDNF in hippocampus and amygdala immediately after contextual conditioning in POT model of PTSD in mice.

MEN1 Tumor Suppressor Gene is Required for Long-term Memory Formation in an Aversive Operant Conditioning Model of Lymnaea stagnalis

Activity-dependent transcription factors critically coordinate the gene expression program underlying memory formation. The tumor suppressor gene, MEN1, encodes a ubiquitously expressed transcription regulator required for synaptogenesis and synaptic plasticity in invertebrate and vertebrate central neurons. In this study, we investigated the role of MEN1 in long-term memory (LTM) formation in an aversive operant conditioning paradigm in the freshwater pond snail Lymnaea stagnalis (L. stagnalis). We demonstrated that LTM formation is associated with an increased expression of MEN1 coinciding with an up-regulation of creb1 gene expression. In vivo knockdown of MEN1 prevented LTM formation and conditioning-induced changes in neuronal activity in the identified pacemaker neuron RPeD1. Our findings suggest the involvement of a new pathway in LTM consolidation that requires MEN1-mediated gene regulation.

Trace Conditioning in Drosophila Induces Associative Plasticity in Mushroom Body Kenyon Cells and Dopaminergic Neurons.

Dopaminergic neurons (DANs) signal punishment and reward during associative learning. In mammals, DANs show associative plasticity that correlates with the discrepancy between predicted and actual reinforcement (prediction error) during classical conditioning. Also in insects, such as Drosophila, DANs show associative plasticity that is, however, less understood. Here, we study associative plasticity in DANs and their synaptic partners, the Kenyon cells (KCs) in the mushroom bodies (MBs), while training Drosophila to associate an odorant with a temporally separated electric shock (trace conditioning). In most MB compartments DANs strengthened their responses to the conditioned odorant relative to untrained animals. This response plasticity preserved the initial degree of similarity between the odorant- and the shock-induced spatial response patterns, which decreased in untrained animals. Contrary to DANs, KCs (α'/β'-type) decreased their responses to the conditioned odorant relative to untrained animals. We found no evidence for prediction error coding by DANs during conditioning. Rather, our data supports the hypothesis that DAN plasticity encodes conditioning-induced changes in the odorant's predictive power.

Electrical Properties of Cementitious Systems: Formation Factor Determination and the Influence of Conditioning Procedures

Abstract The number of people wanting to use electrical tests to determine the transport properties of concrete has increased with advancements in the portability of hand-held testing devices. Electrical measurements are an attractive test method to quantify transport properties of cement-based materials since they can be performed rapidly. There is a high potential for using these tests in quality control or mixture qualification. However, electrical measurements can be significantly influenced by curing and storage conditions, which can impact the degree of saturation, degree of hydration, sample temperature, and pore solution chemistry. This study proposed a general equation that described the electrical resistivity measurements in cementitious systems and possible methods to account for some of these conditioning-induced changes. It is proposed that these tests are useful in the determination of the formation factor, a numerical quantification that describes the microstructure. A comparison of the formation factor obtained from rapid electrical measurements using the Nernst-Einstein relationship was compared to a migration test with the goal of proposing a curing methodology for rapid electrical tests that allows for the determination of a true transport property.

Noradrenergic blockade stabilizes prefrontal activity and enables fear extinction under stress

Stress-induced impairments in extinction learning are believed to sustain posttraumatic stress disorder (PTSD). Noradrenergic signaling may contribute to extinction impairments by modulating medial prefrontal cortex (mPFC) circuits involved in fear regulation. Here we demonstrate that aversive fear conditioning rapidly and persistently alters spontaneous single-unit activity in the prelimbic and infralimbic subdivisions of the mPFC in behaving rats. These conditioning-induced changes in mPFC firing were mitigated by systemic administration of propranolol (10 mg/kg, i.p.), a β-noradrenergic receptor antagonist. Moreover, propranolol administration dampened the stress-induced impairment in extinction observed when extinction training is delivered shortly after fear conditioning. These findings suggest that β-adrenoceptors mediate stress-induced changes in mPFC spike firing that contribute to extinction impairments. Propranolol may be a helpful adjunct to behavioral therapy for PTSD, particularly in patients who have recently experienced trauma.

Deep brain stimulation of the amygdala alleviates fear conditioning-induced alterations in synaptic plasticity in the cortical–amygdala pathway and fear memory

Deep brain stimulation (DBS) of the amygdala has been demonstrated to modulate hyperactivity of the amygdala, which is responsible for the symptoms of post-traumatic stress disorder (PTSD), and thus might be used for the treatment of PTSD. However, the underlying mechanism of DBS of the amygdala in the modulation of the amygdala is unclear. The present study investigated the effects of DBS of the amygdala on synaptic transmission and synaptic plasticity at cortical inputs to the amygdala, which is critical for the formation and storage of auditory fear memories, and fear memories. The results demonstrated that auditory fear conditioning increased single-pulse-evoked field excitatory postsynaptic potentials in the cortical-amygdala pathway. Furthermore, auditory fear conditioning decreased the induction of paired-pulse facilitation and long-term potentiation, two neurophysiological models for studying short-term and long-term synaptic plasticity, respectively, in the cortical-amygdala pathway. In addition, all these auditory fear conditioning-induced changes could be reversed by DBS of the amygdala. DBS of the amygdala also rescued auditory fear conditioning-induced enhancement of long-term retention of fear memory. These findings suggested that DBS of the amygdala alleviating fear conditioning-induced alterations in synaptic plasticity in the cortical-amygdala pathway and fear memory may underlie the neuromodulatory role of DBS of the amygdala in activities of the amygdala.

Fear conditioning-related changes in cerebellar Purkinje cell activities in goldfish.

BackgroundFear conditioning-induced changes in cerebellar Purkinje cell responses to a conditioned stimulus have been reported in rabbits. It has been suggested that synaptic long-term potentiation and the resulting increases in firing rates of Purkinje cells are related to the acquisition of conditioned fear in mammals. However, Purkinje cell activities during acquisition of conditioned fear have not been analysed, and changes in Purkinje cell activities throughout the development of conditioned fear have not yet been investigated. In the present study, we tracked Purkinje cell activities throughout a fear conditioning procedure and aimed to elucidate further how cerebellar circuits function during the acquisition and expression of conditioned fear.MethodsActivities of single Purkinje cells in the corpus cerebelli were tracked throughout a classical fear conditioning procedure in goldfish. A delayed conditioning paradigm was used with cardiac deceleration as the conditioned response. Conditioning-related changes of Purkinje cell responses to a conditioned stimulus and unconditioned stimulus were examined.ResultsThe majority of Purkinje cells sampled responded to the conditioned stimulus by either increasing or decreasing their firing rates before training. Although there were various types of conditioning-related changes in Purkinje cells, more than half of the cells showed suppressed activities in response to the conditioned stimulus after acquisition of conditioned fear. Purkinje cells that showed unconditioned stimulus-coupled complex-spike firings also exhibited conditioning-related suppression of simple-spike responses to the conditioned stimulus. A small number of Purkinje cells showed increased excitatory responses in the acquisition sessions. We found that the magnitudes of changes in the firing frequencies of some Purkinje cells in response to the conditioned stimulus correlated with the magnitudes of the conditioned responses on a trial-to-trial basis.ConclusionsThese results demonstrate that Purkinje cells in the corpus cerebelli of goldfish show fear conditioning-related changes in response to a stimulus that had been emotionally neutral prior to conditioning. Unconditioned stimulus-induced climbing fibre inputs to the Purkinje cells may be involved in mediating these plastic changes.

Effect Of Stage Of Conditioning And Acute Exercise On Insulin Sensitivity In Elite Endurance Athletes.

Fitness-associated sensitivity to insulin is presumably the consequence of regular exercise sessions, but it is unclear what aspect of exercise results in a change in insulin sensitivity. PURPOSE: This study was conducted to test the hypothesis that conditioning-induced changes in insulin sensitivity are a function of relative metabolic stress produced by exercise. METHODS: Eighteen mixed-breed elite racing sled dogs were examined in the early fall (unconditioned), late fall (moderate conditioning) and winter (fully conditioned). At each stage of conditioning, insulin sensitivity was assessed using a euglycemic-hyperinsulinemic clamp before and 60 hr after a standardized exercise challenge (22 miles covered in 2.5 hr). Muscle biopsies (pre-exercise, immediately post-exercise and 60 hr post-exercise) were obtained at the same 3 levels of conditioning to assess acute and chronic biochemical and molecular responses to the standardized exercise challenge. Analysis of variance was conducted to determine the effects of stage of conditioning (untrained, moderately trained and fully trained) and time of EHC (pre vs. post ex), and the interaction between these factors, on glucose infusion rate and muscle biochemical and molecular parameters. RESULTS: Pre-exercise insulin sensitivity was significantly higher in both moderately conditioned and fully conditioned dogs, compared to unconditioned dogs (P < 0.0001). The effect of acute exercise on insulin sensitivity was significant only in the unconditioned dogs (P = 0.011), where insulin sensitivity was decreased following the exercise challenge. CONCLUSIONS: These data support the contention that exercise-induced metabolic stress is associated with training-induced changes in insulin sensitivity. This study was supported by the Diabetes Action Research and Education Foundation and Oklahoma State University Center for Veterinary Health Sciences.

Alterations of excitatory transmission in the lateral amygdala during expression and extinction of fear memory

Understanding the neurophysiology of fear extinction has important implications for the treatment of post-traumatic stress disorders. Here we report that fear conditioning resulted in an increase in AMPA/NMDA ratio as well as depression of paired-pulse facilitation (PPF) in neurons of the lateral nucleus of amygdala. These conditioning-induced changes in synaptic transmission were not affected by extinction training. D-cycloserine (DCS), a partial agonist at the glycine-binding site of the NMDA receptor, facilitated extinction and reversed the increase in AMPA/NMDA ratio without altering the depression of PPF when administered before extinction training. Extinction training, however, significantly increased the frequency and amplitude of miniature inhibitory post-synaptic currents and these effects were unaffected by the DCS treatment. Disruption of AMPA receptor endocytosis with a synthetic peptide containing a short C-terminal sequence of GluR2 (869YKEGYNVYG877, GluR23Y) specifically blocked DCS-induced reversal of AMPA/NMDA ratio and the facilitation of extinction. These results suggest that extinction training mainly increases inhibitory transmission leaving conditioning-induced excitatory association unaltered. DCS does not affect inhibitory transmission but reverses the conditioning-induced post-synaptic memory trace when administered before extinction training.

NMDA NR2A and NR2B receptors in the rostral anterior cingulate cortex contribute to pain-related aversion in male rats

NMDA receptors, which are implicated in pain processing, are highly expressed in forebrain areas including the anterior cingulate cortex (ACC). The ACC has been implicated in the affective response to noxious stimuli. Using a combination of immunohistochemical staining, Western blot, electrophysiological recording and formalin-induced conditioned place avoidance (F-CPA) rat behavioral model that directly reflects the affective component of pain, the present study examined formalin nociceptive conditioning-induced changes in the expressions of NMDA receptor subunits NR1, NR2A, and NR2B in the rostral ACC (rACC) and its possible functional significance. We found that unilateral intraplantar (i.pl.) injection of dilute formalin with or without contextual conditioning exposure markedly increased the expressions of NMDA receptor subunits NR2A and NR2B but not of NR1 in the bilateral rACC. NMDA-evoked currents in rACC neurons were significantly greater in formalin-injected rats than in naïve or normal saline-injected rats. Selectively blocking either NR2A or NR2B subunit in the rACC abolished the acquisition of F-CPA and formalin nociceptive conditioning-induced Fos expression, but it did not affect formalin acute nociceptive behaviors and non-nociceptive fear stimulus-induced CPA. These results suggest that both NMDA receptor subunits NR2A and NR2B in the rACC are critically involved in pain-related aversion. Thus, a new strategy targeted at NMDA NR2A or NR2B subunit might be raised for the prevention of pain-related emotional disturbance.

Activity‐dependent depression of the spike after‐depolarization generates long‐lasting intrinsic plasticity in hippocampal CA3 pyramidal neurons

Persistent plastic changes to the intrinsic excitability of neurons have substantial implications for computational processing within the CNS. We have identified and characterized a novel long-lasting form of intrinsic plasticity in hippocampal CA3 pyramidal cells. Although the patterns of action potential firing elicited in this cell population by depolarizing current injections exhibited considerable diversity, practically all cells produced an initial high frequency (>100 Hz) burst of two to five spikes. This burst involved conductances that were responsible for the prominent spike afterdepolarization of CA3 pyramids. Long-lasting changes in the firing behaviour of CA3 cells were produced by conditioning stimuli (CS) consisting of either periods of depolarization in voltage clamp or periods of short (2 or 4 spikes) high frequency (circa 100 Hz) burst firing at 5 or 10 Hz. CS-induced changes included substantial prolongation of the first inter-spike interval and increased spike jitter. Similar CS-induced changes were seen when the test stimulus used to elicit firing resembled a glutamatergic EPSC. In line with this, a long-lasting depression of the ADP was elicited by the same CS that altered firing patterns of CA3 cells. Conditioning-induced changes in both spiking patterns and ADP amplitude were blocked by buffering intracellular Ca(2+) with BAPTA. Furthermore, the Kv7 channel blocker XE991, a cognitive enhancer, both enhanced the ADP and completely eliminated its conditioning-induced depression. These findings indicate that a persistent enhancement of Kv7 channels, following a transient increase in cytoplasmic Ca(2+), results in a prolonged depression of the ADP in CA3 pyramidal neurones.

Fear Conditioning-Induced Alterations of Phospholipase C-β1a Protein Level and Enzyme Activity in Rat Hippocampal Formation and Medial Frontal Cortex

We investigated the effects of one-trial fear conditioning on phospholipase C-β1a catalytic activity and protein level in hippocampal formation and medial frontal cortex of untreated control rats and rats prenatally exposed to ethanol. One hour following fear conditioning of untreated control rats, phospholipase C-β1a protein level was increased in the hippocampal cytosolic fraction and decreased in the hippocampal membrane and cortical cytosolic and cortical membrane fractions. Twenty-four hours after fear conditioning, phospholipase C-β1a protein level was reduced in the hippocampal cytosolic fraction and elevated in the cortical nuclear fraction; in addition, 24 h after conditioning, phospholipase C-β1a activity in the cortical cytosolic fraction was increased. Rats that were exposed prenatally to ethanol displayed attenuated contextual fear conditioning, whereas conditioning to the acoustic-conditioned stimulus was not different from controls. In behavioral control (unconditioned) rats, fetal ethanol exposure was associated with reduced phospholipase C-β1a enzyme activity in the hippocampal nuclear, cortical cytosolic, and cortical membrane fractions and increased phospholipase C-β1a protein level in the hippocampal membrane and cortical cytosolic fractions. In certain cases, prenatal ethanol exposure modified the relationship between fear conditioning and changes in phospholipase C-β1a protein level and/or activity. The majority of these effects occurred 1 h, rather than 24 h, after fear conditioning. Multivariate analysis of variance revealed interactions between fear conditioning, subcellular fraction, and prenatal ethanol exposure for measures of phospholipase C-β1a protein level in hippocampal formation and phospholipase C-β1a enzyme activity in medial frontal cortex. In the majority of cases, fear conditioning-induced changes in hippocampal phospholipase C-β1a protein level were augmented in rats prenatally exposed to ethanol. In contrast, fear conditioning-induced changes in cortical phospholipase C-β1a activity were, often, in opposite directions in prenatal ethanol-exposed compared to diet control rats. We speculate that alterations in subcellular phospholipase C-β1a catalytic activity and protein level contribute to contextual fear conditioning and that learning deficits observed in rats exposed prenatally to ethanol result, in part, from dysfunctions in phospholipase C-β1a signal transduction.

Cellular Traces of Behavioral Classical Conditioning Can Be Recorded at Several Specific Sites in a Simple Nervous System

We used a behavioral learning paradigm followed by electrophysiological analysis to find sites in the Lymnaea feeding network in which electrical changes could be recorded after appetitive conditioning. Specifically, we analyzed conditioning-induced changes in cellular responses in the mechanosensory conditioned stimulus (CS) pathway, in the central pattern generator (CPG) network, and in feeding motoneurons. During training, experimental animals received 15 pairings of lip touch (the CS) with sucrose (the unconditioned stimulus, US). Control animals received 15 random CS and US presentations. Electrophysiological tests on semi-intact preparations made from conditioned animals demonstrated a network correlate of the overall feeding conditioned response, a touch-evoked CPG-driven fictive feeding rhythm. At the motoneuronal level, we found significant conditioning-induced increases in the amplitude of an early touch-evoked EPSP and spike activity, recorded from the B3 feeding motoneuron. Increases in EPSP amplitude and motoneuronal spike activity could occur independently of conditioned fictive feeding. These changes in response recorded at the level of CPG interneurons, and motoneurons were preceded by changes recorded in the CS pathway. This was demonstrated by recording a conditioning-induced increase in the number of touch-evoked spikes in the cerebrobuccal connective, which forms part of the CS pathway. The finding that electrophysiological changes after conditioning can be recorded at multiple sites in this simple system provided an important intermediate level of analysis between whole animal behavior and cellular studies on the synaptic sites of plasticity.

Classical Conditioning-Induced Changes in Low-Molecular-Weight GTP-Binding Proteins in Rabbit Hippocampus

Classical conditioning of Hermissenda, involving paired light-rotation events, results in a 30-35% decrease in the levels of a 20-kDa G protein (cp20). To test whether a similar protein exists in vertebrates, rabbits were trained to associate a tone with periorbital electrical stimulation and G proteins were analyzed by photoaffinity labeling with [alpha-32P]GTP-azidoanilide. A 20-kDa G protein similar to cp20 decreased by 36% in the hippocampus of rabbits subjected to paired tone and electrical stimulation, but not in unpaired controls. Learning-specific decreases were also found in the amount of ras protein.

Sequential modification of membrane currents with classical conditioning

Pavlovian conditioning of the nudibranch mollusc Hermissenda crassicornis was previously shown to produce long-lasting reduction of two K+ currents measured across the Type B photoreceptor soma membrane (Alkon et al., 1982a; Alkon et al., 1985). Pavlovian conditioning of the rabbit was also shown to be followed by persistent K+ current reduction (Disterhoft et al., 1986). Here we report the first evidence that Ca2+ currents can also be modified by conditioning. The amplitude of the currents rather than their voltage-dependence remains reduced at least 1-2 d after conditioning (but not control procedures). Conditioning-induced changes of both K+ and Ca2+ currents increased as a function of training, the Ca2+ currents only changing substantially with greater than or equal to 250 trials. The later changes of the Ca2+ current may function to limit the magnitude of excitability increases due to associative learning.

Protein Kinase Injection Reduces Voltage-Dependent Potassium Currents

Intracellular iontophoretic injection of the catalytic subunit of cyclic adenosine monophosphate-dependent protein kinase increased input resistance and decreased a delayed voltage-dependent K+ current of the type B photoreceptor in the nudibranch Hermissenda crassicornis to a greater extent than an early, rapidly inactivating K+ current (IA). This injection also enhanced the long-lasting depolarization of type B cells after a light step. These findings suggest the involvement of cyclic adenosine monophosphate-dependent phosphorylation in the differential regulation of photoreceptor K+ currents particularly during illumination. On the other hand, conditioning-induced changes in IA may also be regulated by a different type of phosphorylation (for example, Ca2+-dependent).