Biomedical subjects

B G Schreurs

Publications and source records attributed to B G Schreurs.

At least 19 recordsLinked to original sources

PubMed2003

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↗

PubMed2001

Imaging learning and memory: classical conditioning.

The search for the biological basis of learning and memory has, until recently, been constrained by the limits of technology to classic anatomic and electrophysiologic studies. With the advent of functional imaging, we have begun to delve into what, for many, was a "black box." We review several different types of imaging experiments, including steady state animal experiments that image the functional labeling of fixed tissues, and dynamic human studies based on functional imaging of the intact brain during learning. The data suggest that learning and memory involve a surprising conservation of mechanisms and the integrated networking of a number of structures and processes.

Animals↗

PubMed2001

Gene expression profiles during long-term memory consolidation.

Changes in gene expression have been postulated to occur during long-term memory (LTM). We used high-density cDNA microarrays to assess changes in gene expression 24 h after rabbit eye blink conditioning. Paired animals were presented with a 400 ms, 1000 Hz, 82 dB tone conditioned stimulus that coterminated with a 100 ms, 60 Hz, 2 mA electrical pulse unconditioned stimulus. Unpaired animals received the same conditioned and unconditioned stimuli but presented in an explicitly unpaired manner. Differences in expression levels between paired and unpaired animals in the hippocampus and cerebellar lobule HVI, two regions activated during eye blink conditioning, indicated the involvement of novel genes as well as the participation of previously implicated genes. Patterns of gene expression were validated by in situ hybridization. Surprisingly, the data suggest that an underlying mechanism of LTM involves widespread decreased, rather than increased, gene expression. These results demonstrate the feasibility and utility of a cDNA microarray system as a tool for dissecting the molecular mechanisms of associative memory.

Animals↗

PubMed2001

Conditioning-specific reflex modification of the rabbit (Oryctolagus cuniculus) nictitating membrane response: generality and nature of the phenomenon.

Conditioning-specific reflex modification (CRM) occurs when classical conditioning modifies responding to an unconditioned stimulus in the absence of a conditioned stimulus. This form of reflex modification suggests that learning modifies the unconditioned reflex pathway. Rabbit (Oryctolagus cuniculus) nictitating membrane responses to 5 intensities and 3 durations of airpuff (AP) or periorbital electrical stimulation (ES) were monitored before and after conditioning. AP tests detected strong CRM after conditioning with ES and modest levels of CRM after conditioning with AP. After conditioning with AP, ES tests failed to detect CRM. After conditioning with a stronger AP, CRM was again detected by AP tests. CRM is a general phenomenon but is more readily detected after training with a relatively aversive stimulus; thus, it may be a function of level of arousal.

Amygdala↗

PubMed2000

Conditioning the unconditioned response: modification of the rabbit's (Oryctolagus cuniculus) unconditioned nictitating membrane response.

Conditioning-specific reflex modification (CRM) occurs when classical conditioning modifies responding to an unconditioned stimulus (US) in the absence of a conditioned stimulus (CS). Three experiments monitored rabbit nictitating (Oryctolagus cuniculus) membrane unconditioned responses to 5 intensities and 4 durations of periorbital electrical stimulation before and after CS or US manipulation. CRM occurred after 12 days of CS-US pairings but not following unpaired CS/US presentations or restraint. CRM survived CS-alone and CS/US-unpaired extinction of the conditioned response (CR) but not presentations of the US alone, although CRs remained intact. Thus, CRs could be weakened without eliminating CRM and CRM could be weakened without eliminating CRs. Data indicate CRM is a reliable, associative effect that is more than a generalized CR and may not be explained by habituation, stimulus generalization, contextual conditioning, or bidirectional conditioning.

Adaptation, Physiological↗

PubMed2000

Kinetic and frequency-domain properties of reflex and conditioned eyelid responses in the rabbit.

Eyelid position and the electromyographic activity of the orbicularis oculi muscle were recorded unilaterally in rabbits during reflex and conditioned blinks. Air-puff-evoked blinks consisted of a fast downward phase followed sometimes by successive downward sags. The reopening phase had a much longer duration and slower peak velocity. Onset latency, maximum amplitude, peak velocity, and rise time of reflex blinks depended on the intensity and duration of the air puff-evoking stimulus. A flashlight focused on the eye also evoked reflex blinks, but not flashes of light, or tones. Both delayed and trace classical conditioning paradigms were used. For delayed conditioning, animals were presented with a 350-ms, 90-dB, 600-Hz tone, as conditioned stimulus (CS). For trace conditioning, animals were presented with a 10-ms, 1-k/cm(2) air puff, as CS. The unconditioned stimulus (US) consisted of a 100-ms, 3-k/cm(2) air puff. The stimulus interval between CS and US onsets was 250 ms. Conditioned responses (CRs) to tones were composed of downward sags that increased in number through the successive conditioning sessions. The onset latency of the CR decreased across conditioning at the same time as its maximum amplitude and its peak velocity increased, but the time-to-peak of the CR remained unaltered. The topography of CRs evoked by short, weak air puffs as the CS showed three different components: the alpha response to the CS, the CR, and the reflex response to the US. Through conditioning, CRs showed a decrease in onset latency, and an increase in maximum amplitude and peak velocity. The time-to-peak of the CR remained unchanged. A power spectrum analysis of reflex and conditioned blink acceleration profiles showed a significant approximately 8-Hz oscillation within a broadband of frequencies between 4 and 15 Hz. Nose and mandible movements presented power spectrum profiles different from those characterizing reflex and conditioned blinks. It is concluded that eyelid reflex responses in the rabbit present significant differences from CRs in their profiles and metric properties, suggesting different neural origins, but that a common approximately 8-Hz neural oscillator underlies lid motor performance. According to available data, the frequency of this putative oscillator seems to be related to the species size.

Air Movements↗

PubMed1999

The effects of scopolamine on changes in regional cerebral blood flow during classical conditioning of the human eyeblink response.

We examined the effects of scopolamine on the functional anatomy of classical conditioning of the human eyeblink response. Ten healthy young normal female volunteers (mean age +/- SEM: 26.7 +/- 0.9 years) were administered 0.4 mg scopolamine intravenously 1 h before regional cerebral blood flow (rCBF) was measured with positron emission tomography (PET) and H215O. Scans occurred during three sequential phases: (1) explicitly unpaired presentations of the unconditioned stimulus (airpuff to the right eye) and conditioned stimulus (binaural tone), (2) paired presentations of the two stimuli (associative learning) and (3) explicitly unpaired presentation of the stimuli (extinction phase). Scopolamine impaired acquisition of the conditioned eyeblink response (54.7 +/- 4.9%) relative to 18 untreated subjects from two previous PET studies. Regions that showed significant relative increases in rCBF during conditioning included the right lateral occipital cortex, the right inferior occipital cortex, the right lateral temporo-occipital cortex, the left medial temporo-occipital cortex, the posterior cingulate, the right cerebellum/brain stem area and the medial cerebellum. Significant relative decreases in rCBF were measured in the thalamus, the left putamen/insula area, the right putamen and the left and middle cerebellar cortex. The data partially replicate previous findings in unmedicated young volunteers of conditioning-specific rCBF changes in the cingulate cortex, the cerebellar cortex, the insula and the lateral temporo-occipital cortex. Our finding of decreased rCBF in the thalamus and increased rCBF in the occipital cortex may be attributable to effects of scopolamine per se rather than conditioning. Our data lend further support to the notion that classical conditioning involves distributed changes in multiple systems within the central nervous system.

Acoustic Stimulation↗

PubMed1998

Classical conditioning increases membrane-bound protein kinase C in rabbit cerebellum.

We examined membrane-bound protein kinase C (PKC) in the cerebellum of rabbits given paired presentations of a tone conditioned stimulus (CS) that co-terminated with a periocular electrical stimulation unconditioned stimulus (US) or unpaired presentations of the CS and US or restraint in the experimental context. PKC activation was measured by quantitative film autoradiography of [3H]phorbol 12,13-dibutyrate ([3H]PBt2) binding in the molecular and granule cells layers of lobule HVI, anterior vermis and Crus I, and in the dentate/interpositus nuclei. There was a statistically significant increase in [3H]PBt2 binding within the molecular layer of lobule HVI in rabbits given paired training relative to controls. The results indicate PKC activation in lobule HVI may be important in acquisition of conditioned eyeblink responses.

Animals↗

PubMed1998

Intracellular correlates of acquisition and long-term memory of classical conditioning in Purkinje cell dendrites in slices of rabbit cerebellar lobule HVI.

Intradendritic recordings in Purkinje cells from a defined area in parasaggital slices of cerebellar lobule HVI, obtained after rabbits were given either paired (classical conditioning) or explicitly unpaired (control) presentations of tone and periorbital electrical stimulation, were used to assess the nature and duration of conditioning-specific changes in Purkinje cell dendritic membrane excitability. We found a strong relationship between the level of conditioning and Purkinje cell dendritic membrane excitability after initial acquisition of the conditioned response. Moreover, conditioning-specific increases in Purkinje cell excitability were still present 1 month after classical conditioning. Although dendritically recorded membrane potential, input resistance, and amplitude of somatic and dendritic spikes were not different in cells from paired or control animals, the size of a potassium channel-mediated transient hyperpolarization was significantly smaller in cells from animals that received classical conditioning. In slices of lobule HVI obtained from naive rabbits, the conditioning-related increases in membrane excitability could be mimicked by application of potassium channel antagonist tetraethylammonium chloride, iberiotoxin, or 4-aminopyridine. However, only 4-aminopyridine was able to reduce the transient hyperpolarization. The pharmacological data suggest a role for potassium channels and, possibly, channels mediating an IA-like current, in learning-specific changes in membrane excitability. The conditioning-specific increase in Purkinje cell dendritic excitability produces an afterhyperpolarization, which is hypothesized to release the cerebellar deep nuclei from inhibition, allowing conditioned responses to be elicited via the red nucleus and accessory abducens motorneurons.

Action Potentials↗

PubMed1998

Pairing-specific long-term depression prevented by blockade of PKC or intracellular Ca2+.

Long-term depression was established in cerebellar Purkinje cells using 20 pairings of a brief, high frequency train of parallel fiber stimulation with a subsequent 100 ms depolarizing current injection. Effects were assessed on the peak amplitude of Purkinje cell excitatory post synaptic potentials (EPSPs) elicited by single parallel fiber test pulses. Intradendritically recorded Purkinje cell EPSPs exhibited long-term (>20 min) reduction in peak amplitude following paired stimulation but not if pretreated with the protein kinase C inhibitor calphostin C or iontophoretically injected with the calcium chelator EGTA. The similarity of the stimulation protocol and behavioral conditioning paradigms suggests pairing-specific long-term synaptic depression is a valuable model for understanding the cellular mechanisms underlying cerebellar cortical contributions to learning.

Animals↗

PubMed1997

Dendritic excitability microzones and occluded long-term depression after classical conditioning of the rabbit's nictitating membrane response.

We made intradendritic recordings in Purkinje cells (n = 164) from parasaggital slices of cerebellar lobule HVI obtained from rabbits given paired presentations of tone and periorbital electrical stimulation (classical conditioning, n = 27) or explicitly unpaired presentations of tone and periorbital stimulation (control, n = 16). Purkinje cell dendritic membrane excitability, assessed by the current required to elicit local dendritic calcium spikes, increased significantly in slices from animals that received classical conditioning. In contrast, membrane potential, input resistance, and amplitude of somatic and dendritic spikes were not different in slices from animals given paired or explicitly unpaired stimulus presentations. The location of cells with low thresholds for local dendritic calcium spikes suggested that there are specific sites for learning-related changes within lobule HVI. These areas may correspond to learning "microzones" and are consistent with locations of learning-related in vivo changes in Purkinje cell activity. Application of 4-aminopyridine, an antagonist of the rapidly inactivating potassium current IA, reduced the threshold for dendritic spikes in slices from naive animals to levels found in slices from trained animals. In cells where thresholds for eliciting parallel fiber-stimulated Purkinje cell excitatory postsynaptic potentials (EPSPs) were measured, levels of parallel fiber stimulation required to elicit a 6-mV EPSP as well as a 4-mV EPSP (n = 30) and a Purkinje cell spike (n = 56) were found to be significantly lower in slices from paired animals than unpaired controls. A classical conditioning procedure was simulated in slices of lobule HVI by pairing a brief, high-frequency train of parallel fiber stimulation (8 pulses, 100 Hz) with a brief, lower frequency train of climbing fiber stimulation (3 pulses, 20 Hz) to the same Purkinje cell. Following paired stimulation of the parallel and climbing fibers, Purkinje cell EPSPs underwent a long-term (> 20 min) reduction in peak amplitude (-24%) in cells (n = 12) from animals given unpaired stimulus presentations but to a far less extent (-9%) in cells (n = 20) from animals given in vivo paired training. Whereas 92% of cells from unpaired animals showed pairing-specific depression, 50% of cells from paired animals showed no depression and in several cases showed potentiation. Our data establish that there are localized learning-specific changes in membrane and synaptic excitability of Purkinje cells in rabbit lobule HVI that can be detected in slices 24 h after classical conditioning. Long-term changes within Purkinje cells that effect this enhanced excitability may occlude pairing-specific long-term depression.

Animals↗

PubMed1997

Lateralization and behavioral correlation of changes in regional cerebral blood flow with classical conditioning of the human eyeblink response.

Laterality of changes in regional cerebral blood flow (rCBF) during classical conditioning of the human eyeblink response was studied and changes in rCBF were correlated with conditioned responses. In 10 normal volunteers, rCBF was mapped with positron emission tomography and H2(15)O during pairings of a binaural tone conditioned stimulus and an air puff unconditioned stimulus to the left eye. Control conditions consisted of explicitly unpaired presentations of the tone and air puff before (control) and after (extinction) pairings. During pairings, rCBF increased significantly in right primary auditory cortex (contralateral to air puff) and decreased significantly in left and right cerebellar cortex. There were also increases in rCBF in right auditory association cortex and left temporoccipital cortex. Decreases in rCBF were noted bilaterally in the temporal poles and in the left prefrontal cortex. Positive correlations between changes in rCBF and percent conditioned responses were located in middle cerebellum, right superior temporal cortex, left dorsal premotor cortex, right middle cingulate, and right superior temporal cortex. There were negative correlations in left inferior prefrontal cortex, left middle prefrontal cortex, and right inferior parietal cortex. The data replicate our previous findings of lateralized changes in rCBF following presentations of a binaural tone and air puff to the right eye and indicate that there are pairing-specific changes in primary auditory cortex and cerebellum that are not unique to the left or right hemisphere but are a function of the side of training. The commonalities as well as differences in regional involvement in our present and previous experiment as well as in other eyeblink studies illustrate the advantage of functional neuroimaging to quantify different strategies used by the brain to perform seemingly similar functions. Indeed, the data support the notion that learning-related changes can be detected in a number of specific, but not necessarily invariant, brain regions, and that the involvement of any one region is dependent on the characteristics of the particular learning situation.

Acoustic Stimulation↗

PubMed1996

Calexcitin: a signaling protein that binds calcium and GTP, inhibits potassium channels, and enhances membrane excitability.

A previously uncharacterized 22-kDa Ca(2+)-binding protein that also binds guanosine nucleotides was characterized, cloned, and analyzed by electrophysiological techniques. The cloned protein, calexcitin, contains two EF-hands and also has homology with GTP-binding proteins in the ADP ribosylation factor family. In addition to binding two molecules of Ca2+, calexcitin bound GTP and possessed GTPase activity. Calexictin is also a high affinity substrate for protein kinase C. Application of calexcitin to the inner surface of inside-out patches of human fibroblast membranes, in the presence of Ca2+ and the absence of endogenous Ca2+/calmodulin kinase type II or protein kinase C activity, reduced the mean open time and mean open probability of 115 +/- 6 pS K+ channels. Calexcitin thus appears to directly regulate K+ channels. When microinjected into molluscan neurons or rabbit cerebellar Purkinje cell dendrites, calexcitin was highly effective in enhancing membrane excitability. Because calexcitin translocates to the cell membrane after phosphorylation, calexcitin could serve as a Ca(2+)-activated signaling molecule that increases cellular excitability, which would in turn increase Ca2+ influx through the membrane. This is also the first known instance of a GTP-binding protein that binds Ca2+.

Amino Acid Sequence↗

PubMed1996

High-resolution fluorescent labeling of living cerebellar slices.

In the present study, we extend previous research on staining of living brain slices with fluorescent phospholipids. This new procedure allows high-resolution staining of specific cell types, in particular, Purkinje cells, in the cerebellar slice while not affecting the intrinsic electrical activity of the tissue. Four different nitrobenzoxadiole (NBD)-phospholipids were incorporated into living cerebellar slices via loading from small unilamellar vesicles (SUVs), composed of a carrier and the fluorescent lipid. The labeled acidic phospholipid, NBD-phosphatidic acid (NBD-PA), produced the highest resolution images with exquisite labeling of the dendritic fields. The label was incorporated predominantly into the Purkinje cell body (excluding the nucleus), with more diffuse staining in other cell types, including stellate, basket and granule cells. The labeled lipid concentration and composition of the carrier lipid were significant in determining the specificity of labeling. Labeling, which was optimal after a 1 h incubation, was present throughout the depth of the slice. This procedure provides a promising approach to fluorescent labeling that will allow simultaneous monitoring of changes in cellular morphology and electrophysiology of living brain slices.

4-Chloro-7-nitrobenzofurazan↗

PubMed1996

Pairing-specific long-term depression of Purkinje cell excitatory postsynaptic potentials results from a classical conditioning procedure in the rabbit cerebellar slice.

1. Using a rabbit cerebellar slice preparation, we stimulated a classical conditioning procedure by stimulating parallel fiber inputs to Purkinje cells with the use of a brief, high-frequency train of eight constant-current pulses 80 ms before climbing fiber inputs to the same Purkinje cell were stimulated with the use of a brief, lower frequency train of three constant-current pulses. In all experiments, we assessed the effects of stimulation by measuring the peak amplitude of Purkinje cell excitatory postsynaptic potentials (EPSPs) to single parallel fiber test pulses. 2. Intradendritically recorded Purkinje cell EPSPs underwent a long-term (> 20 min) reduction in peak amplitude (30%) after paired stimulation of the parallel and climbing fibers but not after unpaired or parallel fiber alone stimulation. We call this phenomenon pairing-specific long-term depression (PSD). 3. Facilitation of the peak amplitude of a second EPSP elicited by a parallel fiber train occurred both before and after paired stimulation suggesting that the locus of depression was not presynaptic. Depression of the peak amplitude of a depolarizing response to focal application of glutamate following pairings of parallel and climbing fiber stimulation added support to a suggested postsynaptic locus of the PSD effect. 4. The application of aniracetam potentiated EPSP peak amplitude by 40%, but these values returned to baseline as a result of pairings. With the removal of aniracetam from the bath 20 min after pairings, normal levels of pairing-specific EPSP depression were observed, indicating that the effect did not result from direct desensitization of alpha-amino-3-hydroxy-5-methyl-4-isoxazole-proprionic acid (AMPA) receptors. 5. Incubation of slices in the protein kinase inhibitor H-7 potentiated EPSP peak amplitudes slightly (9%), but peak amplitudes returned to baseline levels after pairings. The net reduction in EPSP peak amplitude of < 10% after pairings suggested that H-7 partially blocked PSD and that, in turn, PSD involved protein kinases. 6. The means of induction and the specificity of those means suggest that the phenomenology of PSD is fundamentally different from that of long-term depression. PSD only occurs with pairings of trains of parallel fiber and climbing fiber stimulation; it occurs without the need for bicuculline; and it can overcome the blocking effects of aniracetam. 7. Nevertheless, the involvement of protein kinases and the potential role of calcium suggest that the mechanisms involved in the induction of PSD and long-term depression have a number of features in common. 8. Because of the pairing-specific nature of the long-term synaptic depression observed in these experiments, PSD provides a mechanism that may contribute to the role of the cerebellar cortex in classical conditioning.

Animals↗

PubMed1995

The effects of scopolamine, lorazepam, and glycopyrrolate on classical conditioning of the human eyeblink response.

Human eyeblink conditioning, a relatively simple form of learning and memory, has previously been shown to be impaired by the central and peripheral anticholinergic scopolamine. The present study compared the behavioral effects of scopolamine with the benzodiazepine lorazepam and a peripherally active anticholinergic, glycopyrrolate. Thirty-six healthy normal volunteers (mean age: 23.7 years) were studied with 12 assigned double-blind to each of three drug conditions (0.5 mg scopolamine IV, 2 mg lorazepam PO, or 0.2 mg glycopyrrolate IV). Subjects underwent classical conditioning of the eyeblink response in which the conditioned stimulus was an 80 dB binaural tone, and the unconditioned stimulus was a 2 psi airpuff to the right eye. Ten trials of unpaired stimulus presentations were followed by 60 paired trials and finally by an extinction period of five tone-alone presentations. An eyeblink response that occurred during the tone but before the airpuff was scored as a conditioned response (CR). Subjects treated with lorazepam (43% mean CRs) and scopolamine (51% mean CRs) exhibited a significantly lower asymptotic level of conditioning than those treated with glycopyrrolate (85% mean CRs; P < 0.01). However, during extinction, lorazepam-treated subjects (35% CRs) showed a lower overall level of responding to the tone than either scopolamine (60% CRs) or glycopyrrolate (62% CRs) treated subjects (P < 0.05). It seems unlikely that these differences could be accounted for by drug-induced alterations in motor responses because there were no significant differences between the three drug conditions in the frequency, latency, or amplitude of unconditioned responses to the airpuff. Overall, our data indicate that scopolamine and lorazepam impair eyeblink conditioning and suggest that some of the effects of benzodiazepines and anticholinergics on learning and memory can be differentiated using this paradigm.

Adult↗

PubMed1995

Conditioning-specific modification of the rabbit's unconditioned nictitating membrane response.

Robust classical conditioning modifies responding to the unconditioned stimulus (US) in the absence of the conditioned stimulus (CS), a phenomenon the researchers called conditioning-specific reflex modification. Unconditioned responses (URs) to periorbital stimulation varying in intensity and duration were assessed before and after 1, 3, or 6 days of paired, explicitly unpaired, or no presentations of tone and electrical stimulation. After 3 days of pairings, conditioned responding (CRs) reached 94%, and there was an increase in latency to the peak of URs. The peak latency increase was replicated in a second experiment where rabbits reached asymptotic conditioning during 6 days of pairings. There was also a conditioning-specific increase in the amplitude of URs. There were no UR changes as a function of low level of CRs following 1 day of pairings. Data suggest that there are learning-specific changes in pathways mediating the US/UR, as well as in those mediating the CS/CR.

Animals↗

PubMed1994

A functional anatomical study of associative learning in humans.

The purpose of the study was to map the functional neuroanatomy of simple associative learning in humans. Eyeblink conditioning was studied in eight normal volunteers using positron emission tomography and H215O. Regional cerebral blood flow was assessed during three sequential phases: (i) explicitly unpaired presentations of the unconditioned stimulus (air puff to the right eye) and conditioned stimulus (binaural tone), (ii) paired presentations of the two stimuli (associative learning), and (iii) presentation of the conditioned stimulus alone. During associative learning, relative to the unpaired phase, blood flow was significantly increased in primary auditory and left posterior cingulate cortices and significantly decreased in areas of the right cerebellar, right prefrontal, right parietal, and insular cortices and right neostriatum. The lateralization of the changes may relate to the functional organization of memory and learning processes in the brain. The activation in primary auditory cortex is an example, using a neuroimaging technique, of a learning-related change in primary sensory cortex in humans. The changes in areas such as the cerebellum, prefrontal cortex, and neostriatum provide support for their roles in associative learning as proposed by animal models. Moreover, these findings show that in humans, even simple classical conditioning involves distributed changes in multiple neural systems.

Adolescent↗