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Classical conditioning and protein kinase C activation regulate the same single potassium channel in Hermissenda crassicornis photoreceptors.

The patch-clamp technique was used to study the effects of classical conditioning and protein kinase C (PKC) activation on K+ channels of identified neurons in the snail Hermissenda crassicornis. Here we present evidence that classical conditioning and PKC activation similarly modify the same K+ channel. K+ channels were recorded in cells from animals with different training experience. The 64-pS K+ channel appeared with significantly lower frequency in the conditioned group compared to the frequencies in control animals (naive and unpaired). In addition, when present, the 64-pS channel exhibited a lower percentage of open time and an increased interval between opening bursts in cells from conditioned animals. The 42-pS K+ channel was observed with about the same frequency in all three groups, and its percentage of open time was invariant, regardless of the animal's experience. Incubation of the photoreceptor with the PKC activator phorbol 12,13-dibutyrate (PDBu) led to a profound decrease in the percentage of open time of the 64-pS K+ channel, from 35.7% in the control group to 2.5% in the PDBu-treated group. The inactive phorbol 4 alpha-phorbol 12-myristate 13-acetate had no effect. The use of the PKC inhibitor H-7 significantly blocked the phorbol effect. Inside-out patches obtained from phorbol preincubated cells likewise showed the same effect of PDBu on K+ channels, but the effect was not observed when phorbol was added after the cell-free patches were obtained from nontreated cells. By contrast, the percentage of open time of the 42-pS K+ channel remained unchanged after phorbol treatment.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

Involvement of the amygdala in classical conditioning of eyeblink response in the rat.

The two-factor theory postulates that classical conditioning proceeds through two stages, which support successive acquisition of emotional and motor responses. Emotional conditioning is thought to facilitate the subsequent acquisition of the motor response. This form of interaction between the two stages of learning can be investigated while considering the central role of the amygdala and the cerebellum in emotional and motor conditioning, respectively. Rats with bilateral lesions of the amygdala or the cerebellar interpositus or intact rats were subjected to a fear conditioning session followed by four eyeblink conditioning sessions. Another group of intact rats was subjected to eyeblink conditioning only. The CS in the fear conditioning session was a 73 dB tone, paired with a 100 dB noise-US. The same CS was paired with a periorbital electroshock-US during eyeblink conditioning. Results showed that fear preconditioning facilitated the subsequent eyeblink conditioning among the intact groups. Amygdaloid lesions abolished this facilitatory effect of fear conditioning. These findings demonstrate that amygdala-mediated emotional conditioning facilitates the subsequent acquisition of cerebellum-mediated motor responses.

Amygdala↗

Purkinje cell number related to rate of classical conditioning.

The cerebellar cortex is normally involved (but not essential) in acquisition of the classically conditioned nictitating membrane response. Cerebellar Purkinje cells probably play a role in acquisition, but these cells are vulnerable to hypoxia, ischemia, and aging. Since large age differences exist in rate of conditioning, we suspected that the number of Purkinje cells in individual rabbits would correlate with learning rate. Purkinje cell counts were undertaken in 12 rabbits aged 3-50 months. The correlation between trials to learning criterion and Purkinje cells was -0.79. A second study used 18 3-month-old rabbits. Even with age held constant, the correlation was -0.60. Individual variation in Purkinje cell number accounts for significant variability in learning rate.

Aging↗

In vitro appetitive classical conditioning of the feeding response in the pond snail Lymnaea stagnalis.

In vitro appetitive classical conditioning of the feeding response in the pond snail Lymnaea stagnalis. J. Neurophysiol. 78: 2351-2362, 1997. An in vitro preparation was developed that allowed electrophysiological analysis of appetitive conditioning of feeding in the model molluscan system, Lymnaea. The network generating the feeding motor program (fictive feeding) is well characterized at the cellular level and consists of identified central pattern generator (CPG) interneurons, motor neurons, and modulatory interneurons. Activation of a modulatory interneuron, the slow oscillator (SO), evokes the three-phase fictive feeding rhythm in the same semi-intact preparations where tactile stimuli can be applied to the lips. By pairing touch as a conditioned stimulus (CS) with stimulation of the SO as an unconditioned stimulus (US), we established an effective in vitro paradigm for appetitive conditioning. Before training, touch to the lips evoked only brief and weak activity in the feeding interneurons and motor neurons. After 6-10 conditioning trials, there was a significant enhancement in the fictive feeding response to CS alone. This was not seen in controls (CS only, US only, random CS and US) and in preparations where there was no initial brief response to touch before conditioning. Direct recordings from the protraction phase N1M interneurons during in vitro conditioning indicated that the enhancement of the fictive feeding is due to an increased activation of these CPG cells by mechanosensory inputs from the lips. We also found that the conditioned response was not due to a facilitated activation of modulatory neurons in the feeding network, such as the SO or the cerebral giant cells (CGCs), because the activity of these cells remained unchanged after conditioning.

Animals↗

Disrupted retention of the classically conditioned nictitating membrane response in rabbits with aluminum-induced neurofibrillary degeneration.

Rabbits underwent classical conditioning of the nictitating membrane response (NMR) to a tone conditioned stimulus and an air puff unconditioned stimulus until they emitted 90 percent or greater conditioned responses (CRs) for two consecutive days. They then received intraventricular injection of aluminum chloride, hydrochloric acid or saline. Ten days post injection they were tested for retention of the conditioned response. Animals injected with saline or hydrochloric acid emitted an average of greater than 90 percent CRs. Aluminum injected rabbits, in contrast, emitted an average of only about 40 percent CRs. This disruption of retention of conditioning in aluminum treated rabbits could not be attributed to deficits in sensory or motor processes or to illness. Neuropathological analysis revealed widespread neurofibrillary tangle formation in the aluminum treated animals. Furthermore, the degree of neurofibrillary degeneration was significantly negatively correlated with retention of the CR. The results are considered in the context of using the rabbit NMR preparation as a model system for studying age-related conditioning disorders.

Aluminum↗

Single-unit analysis of different hippocampal cell types during classical conditioning of rabbit nictitating membrane response.

Extracellular single-unit recordings from neurons in the CA1 and CA3 regions of the dorsal hippocampus were monitored during classical conditioning of the rabbit nictitating membrane response. Neurons were classified as different cell types using response to fornix stimulation (i.e., antidromic or orthodromic activation) and spontaneous firing characteristics as criteria. Results showed that hippocampal pyramidal neurons exhibit learning-related neural plasticity that develops gradually over the course of classical conditioning. The learning-dependent pyramidal cell response is characterized by an increase in frequency of firing within conditioning trials and a within-trial pattern of discharge that correlates strongly with amplitude-time course of the behavioral response. In contrast, pyramidal cell activity recorded from control animals given unpaired presentations of the conditioned and unconditioned stimulus (CS and UCS) does not show enhanced discharge rates with repeated stimulation. Previous studies of hippocampal cellular electrophysiology have described what has been termed a theta-cell (19-21, 45), the activity of which correlates with slow-wave theta rhythm generated in the hippocampus. Neurons classified as theta-cells in the present study exhibit responses during conditioning that are distinctly different than pyramidal cells. theta-Cells respond during paired conditioning trials with a rhythmic bursting; the between-burst interval occurs at or near 8 Hz. In addition, two different types of theta-cells were distinguishable. One type of theta-cell increases firing frequency above pretrial levels while displaying the theta bursting pattern. The other type decreases firing frequency below pretrial rates while showing a theta-locked discharge. In addition to pyramidal and theta-neurons, several other cell types recorded in or near the pyramidal cell layer could be distinguished. One cell type was distinctive in that it could be activated with a short, invariant latency following fornix stimulation, but spontaneous action potentials of such neurons could not be collided with fornix shock-induced action potentials. These neurons exhibit a different profile of spontaneous firing characteristics than those of antidromically identified pyramidal cells. Nevertheless, neurons in this noncollidable category display the same learning-dependent response as pyramidal cells. It is suggested that the noncollidable neurons represent a subpopulation of pyramidal cells that do not project an axon via the fornix but project, instead, to other limbic cortical regions.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

Down regulation of cerebellar memory related gene-1 following classical conditioning.

We have isolated and characterized the mRNA of a mouse gene named cerebellar memory related gene-1, previously found by microarray analysis to be differentially expressed following classical conditioning of the rabbit nictitating membrane response. Quantitative RT-PCR analysis showed a significant reduction in mRNA expression in cerebellar lobule HVI but not in the hippocampus of rabbits that received classical conditioning compared to control rabbits that received either unpaired stimulus presentations or were simply restrained. The mouse mRNA encodes a protein of 485 amino acids that includes different potential post-translational modification sites and five copies of the WD-repeat suggesting involvement in protein-protein interaction and regulatory function. In-situ hybridization experiments show highly localized expression of the transcript in mouse brain with the highest expression levels located in the cerebellum, hippocampus and cortex. Taken together, our results reveal a novel gene encoding a WD-repeat protein that is down-regulated in cerebellar lobule HVI as a result of learning and memory.

Animals↗

Classically conditioned enhancement of hyperinsulinemia in the ob/ob mouse.

The obese (C57BL/6J ob/ob) mouse is a commonly used animal model of non-insulin-dependent diabetes mellitus. Recent experiments have shown that stress hyperglycemia can be classically conditioned in the obese but not in the lean mouse. In the present study, classical conditioning of insulin secretion was attempted in C57BL/6J obese and lean animals. For 21 days, obese and lean mice were exposed to a conditioned olfactory stimulus prior to and during eating. On the 22nd day, blood was sampled for all animals following presentation of the conditioned stimulus; testing was repeated 2 weeks later following an additional 4 days of conditioning. Results indicated an effect of conditioning, with significantly greater plasma insulin for trained than for untrained obese mice. That insulin secretion can be more easily conditioned in the obese mouse suggests that a cholinergic mechanism may be involved in the hyperinsulinemia characteristic of this animal.

Animals↗

Two neuronal systems are involved in a classical conditioning in the rat.

Unit activity of the hippocampus, the centrum-medianum-parafascicularis and medialis dorsalis nuclei of the thalamus, was recorded in chronic rats during a classical conditioning; neocortical electroencephalographic and somatic responses were also recorded. Conditioned unit responses of the different groups of neurons were compared according to the precocity of their appearance, their stability, their latency and the differentiation between the positive (reinforced) and the negative (non-reinforced) conditioned stimuli. Conditioned unit responses of type I hippocampal neurons (probably pyramids) and of neurons located in the centrum-medianum-parafascicularis nucleus did not differentiate between the positive and negative conditioned stimuli; they progressed rapidly, then declined and disappeared. They were contemporary with an initial conditioning stage which was characterized by undifferentiated arousal responses (desynchrony of electroencephalographic activity) to both conditioned stimuli. Conditioned unit responses of type II hippocampal neurons (probably granule cells or interneurons of the fascia dentata) and of neurons located in the medialis dorsalis nucleus progressed slowly and differentiated between the conditioned stimuli during a late conditioning stage which was characterized by the regression of arousal responses and the differentiation of somatic responses. These data strongly suggest that two neuronal systems, each having a different role, are involved in classical conditioning in the rat.

Animals↗

Phosphorylation of mitogen-activated protein kinase by one-trial and multi-trial classical conditioning.

The pathway supporting the conditioned stimulus (CS) is one site of plasticity that has been studied extensively in conditioned Hermissenda. Several signal transduction pathways have been implicated in classical conditioning of this preparation, although the major emphasis has been on protein kinase C. Here we provide evidence for the activation and phosphorylation of a mitogen-activated protein kinase (MAPK) pathway by one-trial and multi-trial conditioning. A one-trial in vitro conditioning procedure consisting of light (CS) paired with the application of 5-HT results in the increased incorporation of 32PO4 into proteins detected with two-dimensional gel electrophoresis. Two of the phosphoproteins have molecular weights of 44 and 42 kDa, consistent with extracellular signal-regulated protein kinases (ERK1 and ERK2). Phosphorylation of the 44 and 42 kDa proteins by one-trial conditioning was inhibited by pretreatment with PD098059, A MEK1 (ERK-Activating kinase) inhibitor. Assays of ERK activity with brain myelin basic protein as a substrate revealed greater ERK activity for the group that received one-trial conditioning compared with an unpaired control group. Western blot analysis of phosphorylated ERK using antibodies recognizing the dually phosphorylated forms of ERK1 and ERK2 showed an increase in phosphorylation after one-trial conditioning compared with unpaired controls. The increased phosphorylation of ERK after one-trial conditioning was blocked by pretreatment with PD098059. Hermissenda that received 10 or 15 conditioning trials showed significant behavioral suppression compared with pseudo-random controls. After conditioning and behavioral testing, the conditioned animals showed significantly greater phosphorylation of ERK compared with the pseudo-random controls. These results show that the ERK-MAPK signaling pathway is activated in Pavlovian conditioning of Hermissenda.

Animals↗

Pharmacological differentiation of allergic and classically conditioned asthma in the guinea pig.

A whole-body plethysmographic technique was developed and then used to detect experimentally induced asthma in guinea pigs and to assess pharmacological treatments of allergic and classically conditioned attacks. Inhalation of a beta adrenergic compound (isoproterenol) controlled both forms of attack. Atropine and methscopolamine, parasympathetic blocking agents, prevented conditional but not allergic attacks; diphenhydramine, an antihistamine, prevented allergic attacks; and methysergide, which blocks serotonin (which is believed to trigger human asthma), prevented neither. The guinea pig's allergic reaction is probably the result of a bronchospasm induced by histamine released in tissue of the airway by a local combination of allergen and antibody. The conditional attack is believed to be a constriction of the airway mediated by parasympathetic fibers of central origin.

Animals↗

Sodium pentobarbital: sensory and associative effects in classical conditioning of the rabbit nictitating membrane response.

Four experiments were conducted to determine the effects of sodium pentobarbital (0, 3, 9, and 15 mg/kg) on the acquisition of the rabbit's classically conditioned nictitating membrane response (NMR) and to determine the locus of the drug's effects on sensory, motor, associative, and nonassociative processes. In experiment 1, classical conditioning of the NMR was accomplished by pairing tone and light conditioned stimuli (CSs) with paraorbital shock as the unconditioned stimulus (US). The experiment revealed that pentobarbital retarded the acquisition of conditioned responses (CRs) to both tone and light CSs. Experiment 2, employing unpaired CS, UCS presentations, indicated small but significant drug effects on NMR base rate and nonassociative NMRs to the CS. Experiment 3 revealed no significant drug effect on the psychophysical functions relating UCS intensity to UCR frequency or amplitude, nor on the UCS intensity threshold for eliciting UCRs. On the other hand, in experiment 4, the drug significantly impaired CR frequency over an extended range of CS intensities and raised CS intensity threshold. It was concluded that pentobarbital's attenuation of CS intensity also operated to impair CR acquisition.

Acoustic Stimulation↗

Biochemical studies of stimulus convergence during classical conditioning in Aplysia: dual regulation of adenylate cyclase by Ca2+/calmodulin and transmitter.

Activity-dependent facilitation is a mechanism of associative synaptic plasticity that contributes to classical conditioning in Aplysia. Previous studies of activity-dependent facilitation in the mechanosensory neurons of Aplysia suggested that the Ca2+ influx during paired spike activity enhances the transmitter-stimulated, cAMP-dependent, presynaptic facilitation in these cells. Moreover, paired activity was found to potentiate the activation of the adenylate cyclase by transmitter. It was therefore proposed that the Ca2+/calmodulin-sensitive cyclase may serve as a site of interaction between the inputs from the conditioned and unconditioned stimuli. These studies were carried out to test whether a Ca2+/calmodulin-sensitive adenylate cyclase in the Aplysia CNS has the properties necessary to mediate such an associative interaction. Three lines of evidence indicate that the same cyclase molecules that are sensitive to Ca2+/calmodulin are also stimulated by receptor to facilitatory transmitter via the stimulatory G-protein, Gs: First, calmodulin inhibitors reduced stimulation of the cyclase by facilitatory transmitter. When membranes had been preexposed to one of these inhibitors, trifluoperazine, the addition of exogenous calmodulin partially reversed the inhibition. Second, when Gs had been activated by GTP gamma S, so that it persistently activated the catalytic unit of the cyclase, stimulation of the cyclase by Ca2+ was greatly amplified, suggesting that the two inputs interact in activating a common population of the enzyme. Third, solubilized cyclase activity that bound to calmodulin-Sepharose in a Ca(2+)-dependent manner was stimulated by Gs, which had been partially purified from Aplysia CNS, as well as by Ca2+/calmodulin. Having demonstrated dual activation of the cyclase, we have explored the dependence of cyclase activation on the temporal pattern of Ca2+ and transmitter addition. Optimal activation required that a pulse of Ca2+ temporally overlap the addition of facilitatory transmitter. These several results suggested that the dually regulated adenylate cyclase might underlie the temporal requirements for effective classical conditioning in this system.

Adenylyl Cyclase Inhibitors↗

Impairment of eyeblink classical conditioning in progressive supranuclear palsy.

In a previous study we showed that learning in eyeblink classical conditioning (EBCC) is normal in Parkinson's disease (PD) and that the serial reaction time task (SRTT) is only marginally impaired. Since pathological lesions are more widespread in the atypical parkinsonian disorder of progressive supranuclear palsy (PSP) than in PD, we hypothesized that PSP patients may show more profound deficits in the EBCC and SRTT learning tasks. We therefore investigated EBCC with a delay and two trace paradigms, an SRTT and the California Verbal Learning Test (CVLT) in eight patients with PSP and an age-matched control group. In all EBCC paradigms, we found a significant difference between groups with no significant learning in PSP patients. In the SRTT, implicit learning may have been impaired, but verbal and manual sequence recall were only marginally impaired. Verbal memory was significantly worse in PSP patients than in the control group. Our study shows a dissociated pattern of learning abilities in PSP, where the EBCC as a measure of implicit learning is impaired, the explicit sequence detection in the SRTT is relatively preserved, and the verbal memory impaired. We hypothesize that the PSP patients' deficits in EBCC learning may be due to lesions of deep cerebellar nuclei. There may be a clinical role for EBCC in distinguishing PD and PSP patients.

Aged↗

Cerebellar Purkinje cell activity related to the classically conditioned nictitating membrane response.

Because of the purported critical role of cerebellar lobule HVI in classical conditioning of the nictitating membrane response of the rabbit, we recorded extracellularly from HVI Purkinje cells (PCs) during differential conditioning. Rabbits were trained using tonal conditioned stimuli (CSs) and stimulation of the periocular region as the unconditioned stimulus (US). Many PCs responded to the US, the most frequently observed response being a burst of simple spikes. PCs in HVI showed a variety of responses to CSs that were related to conditioned responses (CRs). The most frequently observed response was an increase in simple spikes correlated with CRs. The activity of many of these cells antedated CRs by 20-200 ms. A smaller proportion of cells exhibited inhibition of simple spike activity that antedated CRs. The existence of PCs that alter their firing before CRs suggests that they may be causally involved in this behavior, and in this respect they reinforce reports that lesions of HVI or its connections disrupt nictitating membrane CRs. Although complex spike activity was not generally related to the US or to CRs, a few PCs responded in relation to CRs with only complex spikes. In demonstrating CR-related activity in cerebellar PCs, this study supports theories of cerebellar learning such as those of Marr and Albus.

Action Potentials↗

Simple neural models of classical conditioning.

A systematic study of the necessary and sufficient ingredients of a successful model of classical conditioning is presented. Models are constructed along the lines proposed by Gelperin, Hopfield, and Tank, who showed that many conditioning phenomena could be reproduced in a model using non-trivial distributed representations of the sensory stimuli. The additional phenomena of extinction and blocking are found to be obtainable by generalizing the Hebbian learning algorithm, rather than by additional complications in the hardware. The most successful algorithms have a minimal number of adjustable parameters, and require only local-time information about the level of postsynaptic activity. The proper behavior of these algorithms is verified by both simple analytic arguments and by direct numerical simulation. Certain detailed assumptions concerning the distributed sensory representations are also found to have a surprising degree of importance.

Algorithms↗

Tracking short-term auditory cortical plasticity during classical conditioning using frequency-tagged stimuli.

Animal studies indicate that short-term plasticity during classical conditioning is a fast process. The temporal details of this process in humans are unknown. We employed amplitude-modulated tones in order to elicit the steady-state field (SSF). Conditioned stimulus (CS+) and CS- had a common low carrier frequency, however, differed in their high-frequency component. Low and high frequencies within one tone were modulated at 29 and 45 Hz, respectively. Mean fast Fourier transformation analysis of each single trial allowed extraction of the cortical response to these modulation frequencies, allowing to track cortical responses trial by trial. Mutilation pictures were used as unconditioned stimulus. Furthermore, heart rate and contingency awareness were assessed. Our main findings are the following: 1) A rapid (within 5 trials) enhancement of the amplitude of the high frequencies in contrast to the low frequency, while the high frequencies differentiated later (toward end of acquisition). This partially replicates rapid plasticity as shown before in animals. 2) Those participants who were less aware of the stimulus contingencies showed a relative heart rate acceleration and greater SSF increase to the CS+. This could possibly imply a stronger early amygdala activation in these participants, which then mediates the development of conditioning-related reorganization in auditory cortical areas.

Acoustic Stimulation↗

Effect of morphine on acquisition of the classically conditioned nictitating membrane response of the rabbit.

The nictitating membrane response of the rabbit was classically conditioned to tone and light-conditioned stimuli presented for 800 msec before delivery of the unconditioned stimulus, consisting of a 100-msec electrical shock to the skin over the paraorbital region of the head. Morphine significantly retarded (1 and 5 mg/kg) or completely blocked (10 mg/kg) the acquisition of conditioned responses. The retarded or blocked acquisition of conditioned responses produced by morphine could still be detected when the rabbits were tested 5 days after cessation of drug injections, suggesting that morphine was affecting acquisition and not performance of conditioned responses. Separate groups of rabbits received explicitly unpaired presentations of tone, light and shock stimuli. The frequency of base-line responding or responding to the tone and light stimuli was low for vehicle controls (1-3%) and was not affected by any dose of morphine. Morphine also had no effect on the latency or amplitude of the unconditioned response to the shock stimulus. These results indicate that the acquisition of conditioned responses was associative, i.e., only occurred during the paired presentations of the conditioned and unconditioned stimuli. Consequently, the retarded acquisition of conditioned responses produced by morphine would appear to be due to an effect on associative learning.

Acoustic Stimulation↗