Acute high-intensity noise induces rapid Arc protein expression but fails to rapidly change GAD expression in amygdala and hippocampus of rats: Effects of treatment with D-cycloserine.

Tinnitus is a devastating auditory disorder impacting a growing number of people each year. The aims of the current experiment were to assess neuronal mechanisms involved in the initial plasticity after traumatic noise exposure that could contribute to the emergence of tinnitus and to test a potential pharmacological treatment to alter this early neural plasticity. Specifically, this study addressed rapid effects of acute noise trauma on amygdalo-hippocampal circuitry, characterizing biomarkers of both excitation and inhibition in these limbic regions, and compared them to expression of these same markers in primary auditory cortex shortly after acute noise trauma. To assess excitatory plasticity, activity-regulated cytoskeleton-associated (Arc) protein expression was evaluated in male rats 45 min after bilateral exposure to acute high-intensity noise (16 kHz, 115 dB SPL, for 1 h), sufficient to cause acute cochlear trauma, a common cause of tinnitus in humans and previously shown sufficient to induce tinnitus in rat models of this auditory neuropathology. Western blot analyses confirmed that up-regulation of amygdalo-hippocampal Arc expression occurred rapidly post-noise trauma, corroborating several lines of evidence from our own and other laboratories indicating that limbic brain structures, i.e. outside of the classical auditory pathways, exhibit plasticity early in the initiation of tinnitus. Western blot analyses revealed no noise-induced changes in amygdalo-hippocampal expression of glutamate decarboxylase (GAD), the biosynthetic enzyme required for GABAergic inhibition. No changes in either Arc or GAD protein expression were observed in primary auditory cortex in this immediate post-noise exposure period, confirming other reports that auditory cortical plasticity may not occur until later in the development of tinnitus. As a further control, our experiments compared Arc protein expression between groups exposed to the quiet background of a sound-proof chamber to those exposed not only to the traumatic noise described above, but also to an intermediate, non-traumatic noise level (70 dB SPL) for the same duration in each of these three brain regions. We found that non-traumatic noise did not up-regulate Arc protein expression in these brain regions. To see if changes in Arc expression due to acute traumatic noise exposure were stress-related, we compared circulating serum corticosterone in controls and rats exposed to traumatic noise at the time when changes in Arc were observed, and found no significant differences in this stress hormone in our experimental conditions. Finally, the ability of D-cycloserine (DCS; an NMDA-receptor NR1 partial agonist) to reduce or prevent the noise trauma-related plastic changes in the biomarker, Arc, was tested. D-cycloserine prevented traumatic noise-induced up-regulation of Arc protein expression in amygdala but not in hippocampus, suggesting that DCS alone is not fully effective in eliminating regionally-specific early plastic changes after traumatic noise exposure.

Memory-enhancing intra-basolateral amygdala clenbuterol infusion reduces post-burst afterhyperpolarizations in hippocampal CA1 pyramidal neurons following inhibitory avoidance learning.

Activation of the basolateral amygdala can modulate the strength of fear memories, including those in single-trial inhibitory avoidance (IA) tasks. Memory retention, measured by the latency to re-enter a dark-compartment paired 24h earlier with a footshock, varies with intensity of this aversive stimulus. When higher intensity footshocks were used, hippocampal CA1 pyramidal neurons exhibited reduced afterhyperpolarizations (AHPs) 24h post-trial, an effect blocked by immediate post-trial inactivation of the basolateral complex of the amygdala (BLA). Similar AHP reductions in CA1 have been observed in a number of learning tasks, with time courses appropriate to support memory consolidation. When less intense footshocks were used for IA training of Sprague-Dawley rats, immediate post-trial infusion of the ß-adrenergic agonist clenbuterol into BLA was required to enhance hippocampal Arc protein expression 45 min later and to enhance memory retention tested 48 h later. Here, using Long-Evans rats and low-intensity footshocks, we confirmed that bilateral immediate post-trial infusion of 15 ng/0.5 µl of the ß-adrenergic agonist clenbuterol into BLA significantly enhances memory for an IA task. Next, clenbuterol was infused into one BLA immediately post-training, with vehicle infused into the contralateral BLA, then hippocampal CA1 neuron AHPs were assessed 24 h later. Only CA1 neurons from hemispheres ipsilateral to post-trial clenbuterol infusion showed learning-dependent AHP reductions. Excitability of CA1 neurons from the same trained rats, but from the vehicle-infused hemispheres, was identical to that from untrained rats receiving unilateral clenbuterol or vehicle infusions. Peak AHPs, medium and slow AHPs, and accommodation were reduced only with the combination of IA training and unilateral BLA ß-receptor activation. Similar to previous observations of BLA adrenergic memory-related enhancement of Arc protein expression in hippocampus, increased CA1 neuronal excitability in the fear-modulated IA task was activated by immediate post-trial ß-receptor activation of the ipsilateral BLA.

D-cycloserine enhances both intrinsic excitability of CA1 hippocampal neurons and expression of activity-regulated cytoskeletal (Arc) protein.

The interaction of NMDA receptor (NMDAR) activation and other mechanisms regulating neuronal excitability have not been thoroughly described. While excess activation of NMDARs results in excitotoxicity, partial activation of NMDARs by d-cycloserine (DCS) is nootropic, enhancing both acquisition and extinction of memories. The mechanism by which DCS treatment enhances memory is unclear. NMDAR activation has been shown to increase expression of the activity-regulated cytoskeletal (Arc) protein associated with neural plasticity and enhanced memory. Enhanced memory is also associated with increases in neuronal intrinsic excitability, i.e. reductions in post-burst afterhyperpolarizations (AHPs) after acquisition of new tasks. Reductions in AHPs can occur when Ca(2+) influx is reduced. This study aimed to determine if either if Arc expression, intrinsic excitability, or both were altered following systemic administration of a memory-enhancing dose of DCS, i.e. what form of plasticity would be exhibited. Both Arc protein expression and intrinsic excitability were enhanced in tissue prepared 1h post-administration of a nootropic dose of DCS. Both mechanisms have been strongly associated with memory enhancement, but have not previously been demonstrated to change across the same time frame in the same preparation in response to DCS treatment.

Fear conditioning alters neuron-specific hippocampal place field stability via the basolateral amygdala.

It is well established that physical changes to an environment result in plasticity of hippocampal place cell activity, while in the absence of changes, place fields are remarkably stable. Manipulations of a rat's perception of the environment without physically changing the environment also result in plasticity of place cell firing. Here, we tested the hypothesis that a rat's perception of an environment could be changed by introducing an auditory fear-conditioned stimulus (CS) to a previously neutral environment, inducing plasticity of hippocampal place fields. First, stable place fields were isolated for rats exploring a radial-arm maze in one environment, and then the rats were fear-conditioned to an auditory CS in a completely separate environment. Later, the CS was specifically paired once with a location in the previously neutral radial-arm maze, either within the given neuron's place field (in-field) or an area outside of the place field (out-of-field). A single, paired presentation of the CS with a location in-field for a specific place cell disrupted the stability of that neuron's place field, whereas pairing the CS with a location out-of-field did not affect place field stability. We further showed that this place field disruption for a CS presented in-field was mediated by inputs from the basolateral amygdala (BLA). Temporarily inactivating the BLA immediately post-CS re-exposure attenuated the CS-induced place field destabilization. Our results show neuron-specific conditional plasticity for actively firing hippocampal place cells, and that the BLA mediates this plasticity when an emotionally arousing or fear-related CS is used.

Acute high-intensity sound exposure alters responses of place cells in hippocampus.

Overstimulation is known to activate neural plasticity in the auditory nervous system causing changes in function and re-organization. It has been shown earlier that overstimulation using high-intensity noise or tones can induce signs of tinnitus. Here we show in studies in rats that overstimulation causes changes in the way place cells of the hippocampus respond as rats search for rewards in a spatial maze. In familiar environments, a subset of hippocampal pyramidal neurons, known as place cells, respond when the animal moves through specific locations but are relatively silent in others. This place-field activity (i.e. location-specific firing) is stable in a fixed environment. The present study shows that activation of neural plasticity through overstimulation by sound can alter the response of these place cells. Rats implanted with chronic drivable dorsal hippocampal tetrodes (four microelectrodes) were assessed for stable single-unit place-field responses that were extracted from multiunit responses using NeuroExplorer computer spike-sorting software. Rats then underwent either 30 min exposure to a 4 kHz tone at 104 dB SPL or a control period in the same sound chamber. The place-field activity was significantly altered after sound exposure showing that plastic changes induced by overstimulation are not limited to the auditory nervous system but extend to other parts of the CNS, in this case to the hippocampus, a brain region often studied in the context of plasticity.

Increased excitability of aged rabbit CA1 neurons after trace eyeblink conditioning.

Cellular properties of CA1 neurons were studied in hippocampal slices 24 hr after acquisition of trace eyeblink conditioning in young adult and aging rabbits. Aging rabbits required significantly more trials than young rabbits to reach a behavioral criterion of 60% conditioned responses in an 80 trial session. Intracellular recordings revealed that CA1 neurons from aging control rabbits had significantly larger, longer lasting postburst afterhyperpolarizations (AHPs) and greater spike frequency adaptation (accommodation) relative to those from young adult control rabbits. After learning, both young and aging CA1 neurons exhibited increased postsynaptic excitability compared with their respective age-matched control rabbits (naive and rabbits that failed to learn). Thus, after learning, CA1 neurons from both age groups had reduced postburst AHPs and reduced accommodation. No learning-related differences were seen in resting membrane potential, membrane time constant, neuron input resistance, or action potential characteristics. Furthermore, comparisons between CA1 neurons from trace-conditioned aging and trace-conditioned young adult rabbits revealed no statistically significant differences in postburst AHPs or accommodation, indicating that similar levels of postsynaptic excitability were attained during successful acquisition of trace eyeblink conditioning, regardless of rabbit age. These data represent the first in vitro demonstration of learning-related excitability changes in aging rabbit CA1 neurons and provide additional evidence for involvement of changes in postsynaptic excitability of CA1 neurons in both aging and learning.

Metrifonate increases neuronal excitability in CA1 pyramidal neurons from both young and aging rabbit hippocampus.

The effects of metrifonate, a second generation cholinesterase inhibitor, were examined on CA1 pyramidal neurons from hippocampal slices of young and aging rabbits using current-clamp, intracellular recording techniques. Bath perfusion of metrifonate (10-200 microM) dose-dependently decreased both postburst afterhyperpolarization (AHP) and spike frequency adaptation (accommodation) in neurons from young and aging rabbits (AHP: p < 0.002, young; p < 0.050, aging; accommodation: p < 0.024, young; p < 0.001, aging). These reductions were mediated by muscarinic cholinergic transmission, because they were blocked by addition of atropine (1 microM) to the perfusate. The effects of chronic metrifonate treatment (12 mg/kg for 3 weeks) on CA1 neurons of aging rabbits were also examined ex vivo. Neurons from aging rabbits chronically treated with metrifonate had significantly reduced spike frequency accommodation, compared with vehicle-treated rabbits. Chronic metrifonate treatment did not result in a desensitization to metrifonate ex vivo, because bath perfusion of metrifonate (50 microM) significantly decreased the AHP and accommodation in neurons from both chronically metrifonate- and vehicle-treated aging rabbits. We propose that the facilitating effect of chronic metrifonate treatment on acquisition of hippocampus-dependent tasks such as trace eyeblink conditioning by aging subjects may be caused by this increased excitability of CA1 pyramidal neurons.

Enhanced synaptic transmission in CA1 hippocampus after eyeblink conditioning.

CA1 field potentials evoked by Schaffer collateral stimulation of hippocampal slices from trace-conditioned rabbits were compared with those from naive and pseudo-conditioned controls. Conditioned rabbits received 80 trace conditioning trials daily until reaching a criterion of 80% conditioned responses in a session. Hippocampal slices were prepared 1 or 24 h after reaching criterion (for trace-conditioned animals) or after a final unpaired stimulus session (for pseudo-conditioned animals); naive animals were untrained. Both somatic and dendritic field potentials were recorded in response to various stimulus durations. Recording and data reduction were performed blind to the conditioning state of the rabbit. The excitatory postsynaptic potential slope was greater in slices prepared from trace-conditioned animals killed 1 h after conditioning than in naive and pseudo-conditioned controls (repeated-measures analysis of variance, F = 4.250, P < 0.05). Associative learning specifically enhanced synaptic transmission between CA3 and CA1 immediately after training. This effect was not evident in the population field potential measured 24 h later.

N-methyl-D-aspartate receptors in associative eyeblink conditioning: both MK-801 and phencyclidine produce task- and dose-dependent impairments.

The effects of N-methyl-D-aspartate receptor blockade on two major variants of rabbit eyeblink conditioning were evaluated using a selective noncompetitive antagonist, [5R, 10S]-[+]-5-methyl-10, 11-dihydro-5H-dibenzo[a, d] cyclo-hepten-5,10-imine hydrogen maleate; dizocilpine (MK-801) or phencyclidine (PCP), a drug of abuse. Either MK-801 or PCP (given daily) impaired rabbits' ability to associate tone conditioned stimuli with airpuff unconditioned stimuli, with the severity of impairment exhibiting clear dose and task dependencies. Trace-conditioned rabbits given > or = 80 micrograms/kg of MK-801 or > or = 1.0 mg/kg of PCP failed to reach a criterion of 80% conditioned responses during training, with significant impairments seen at intermediate doses. Delay-conditioned rabbits, although dose-dependently slowed, successfully acquired the task, even when given doses of MK-801 or PCP that completely blocked acquisition in trace conditioning. Additionally, even low doses of MK-801 (10 micrograms/kg) or of PCP (0.1 mg/kg) severely altered conditioned response timing in trace but not in delay conditioning, resembling effects observed after hippocampal lesions. Doses of MK-801 or PCP that impaired acquisition also severely impaired extinction of both trace- and delay-conditioned eyeblink responses. However, neither MK-801 nor PCP altered retention or timing of previously learned responses. Higher doses of MK-801 (> or = 200 micrograms/kg) or of PCP (> or = 2.0 mg/kg) dose-dependently impaired unconditioned response performance, although lower doses of MK-801 (< or = 160 micrograms/kg) or of PCP (< or = 1.0 mg/kg) had no effects on unconditioned responses or on non-associative pseudoconditioned responses. The deficits observed indicate that although not necessary for retention, N-methyl-D-aspartate receptor activation may facilitate acquisition of delay-conditioning. N-methyl-D-aspartate receptor activation appears to be necessary for acquisition of trace conditioning, and for extinction in either paradigm.

Metrifonate treatment enhances acquisition of eyeblink conditioning in aging rabbits.

The cholinergic system is known to show deterioration during aging and Alzheimer's disease. In response, a therapeutic approach to Alzheimer's disease has been to attempt to compensate for the decrease in central cholinergic function by potentiating the activity of the remaining intact cholinergic cells with cholinesterase inhibitors. In this study treatment with the long-lasting cholinesterase inhibitor metrifonate enhanced acquisition of eyeblink conditioning in aging rabbits without producing interfering side effects. The effects of metrifonate on central and peripheral cholinesterase activity were evaluated, as was the involvement of plasma atropine esterase activity on the central and peripheral response to metrifonate. Results demonstrate that metrifonate can produce predictable, dose-dependent ChE inhibition. Associative learning in the aging rabbit was improved by metrifonate-induced steady state ChE inhibition within a range of 30-80%. Metrifonate was behaviorally effective in the absence of the severe side effects which typically plague cholinesterase inhibitors, suggesting that metrifonate is a possible treatment for the cognitive deficits resulting from normal aging and Alzheimer's disease.

Age- and dose-dependent facilitation of associative eyeblink conditioning by D-cycloserine in rabbits.

Normal aging selectively impairs some forms of learning. For example, aging rabbits require more than twice as many trials to acquire 500-ms trace eyeblink conditioning than do young rabbits. N-methyl-D-aspartate (NMDA) receptor antagonists also impair trace conditioning. The effects of daily D-cycloserine (DCS; a partial agonist of the NMDA receptor-glycine site) treatment were tested on trace conditioning of young or aging rabbits using a conservative quantitative approach. DCS dose dependently improved acquisition, maximally reducing trials to criterion by approximately 50%. Dose-response curves were right-shifted by aging (twice the dose was required to achieve the same enhancement compared with controls). DCS did not affect nonassociative performance but sharpened the conditioned stimulus tone intensity discrimination. DCS thus can functionally modulate NMDA receptors in normal aging, enhance associative learning at all ages, and reduce or reverse age-dependent learning deficits.

Transient changes in excitability of rabbit CA3 neurons with a time course appropriate to support memory consolidation.

1. The excitability of CA3 pyramidal neurons was assessed with intracellular recordings in hippocampal slices from behaviorally naive rabbits. CA3 pyramidal neurons had large (-13.1 +/- 0.3 mV; mean +/- SE) postburst afterhyperpolarization (AHPs) and exhibited robust spike-frequency adaptation (accommodation) to prolonged (800-ms) depolarizing current injection at resting potentials of -68 mV. AHP and accommodation measures differed in scale but not in kind from those obtained in stable recordings from CA1 pyramidal neurons in the same slices or from the same rabbits, with CA3 neurons having larger longer AHPs but fewer spikes during accommodation. 2. Groups of rabbits were trained in a simple, associative-learning task, trace eye-blink conditioning, which required an intact hippocampus for successful acquisition. Memory consolidation in this task also involves the hippocampus, whereas long-term retention of the learned response does not. The time course and magnitude of learning-specific changes in excitability were assessed in 201 CA3 pyramidal neurons. 3. Learning increased the excitability of CA3 pyramidal neurons soon after acquisition (within 1-24 h). The mean postburst AHP was reduced to approximately half (-6.4 +/- 0.3 mV) the basal amplitude of the AHP observed in naive controls. The area and duration of the postburst AHP similarly were reduced. Approximately half of all pyramidal neurons tested soon after learning exhibited significantly reduced AHPs, whereas none exhibited enhanced AHPs. 4. Trace conditioning also reduced accommodation of CA3 pyramidal neurons 1-24 h after learning. Neurons from successfully trained rabbits fired significantly more action potentials (5.6 +/- 1.5) in response to prolonged depolarization than did neurons from naive controls (4.1 +/- 0.2). The magnitude of the learning-specific change in accommodation was less than that for the AHP. Approximately 45% of neurons tested exhibited significantly reduced accommodation soon after learning. 5. Both learning-specific changes in CA3 increased neuronal excitability. Both changes were highly time dependent. AHPs were reduced maximally 1-24 h after learning, then increased, returning to basal (naive) levels within 7 days and remaining basal thereafter. The decay rate of accommodation to basal levels preceded that of the AHP by several days. 6. Other membrane properties, including action potential characteristics, resting potential, and input resistance, were unchanged by learning. The restriction of the observed changes to two interrelated measures of excitability concurs with earlier reports that learning-specific changes in the mammalian hippocampus are linked to changes in a limited number of membrane conductances. 7. Learning, not long-term memory or performance of the learned behavior, was linked to the excitability changes. Neurons from rabbits that failed to acquire the task after considerable training exhibited no excitability changes. Neurons from pseudoconditioned rabbits were indistinguishable from neurons of behaviorally naive controls. Finally, neurons from rabbits that explicitly demonstrated long-term retention of the conditioned response were indistinguishable from those of naive controls. 8. Behavioral changes persisted for extremely long periods, but the observed changes in hippocampal excitability were transient and greatest soon after learning. Excitability was enhanced for a period of a few days, a period demonstrated in other eyeblink studies to be required for memory consolidation. Because hippocampal excitability then returned to basal levels but memory of the learned task persisted, postconsolidation memory traces (the "engram") must be extrahippocampal.

Trace eyeblink conditioning increases CA1 excitability in a transient and learning-specific manner.

Time-dependent, learning-related changes in hippocampal excitability were evaluated by recording from rabbit CA1 pyramidal neurons in slices prepared at various times after acquisition of trace eyeblink conditioning. Increased excitability (reduced postburst afterhyperpolarizations and reduced spike-frequency adaptation) was seen as early as 1 hr after acquisition to behavioral criterion, was maximal in neurons studied 24 hr later, and returned to baseline within 7 d, whereas behavioral performance remained asymptotic for months. Neurons were held at -67 mV to equate voltage-dependent effects. No learning-related effects were observed on input resistance, action-potential amplitude or duration, or resting membrane potential. The excitability changes were learning-specific, because they were not seen in neurons from very slow learning (exhibited < 30% conditioned responses after 15 training sessions) or from pseudoconditioned control rabbits. Neurons from rabbits that displayed asymptotic behavioral performance after long-term retention testing (an additional training session 14 d after learning) were also indistinguishable from control neurons. Thus, the increased excitability of CA1 neurons was not performance- or memory-dependent. Rather, the time course of increased excitability may represent a critical window during which learning-specific alterations in postsynaptic excitability of hippocampal neurons are important for consolidation of the learned association elsewhere in the brain.

Trace eyeblink conditioning in rabbits demonstrates heterogeneity of learning ability both between and within age groups.

Rabbits 2 to 41 months of age were conditioned in the 500 ms trace eyeblink paradigm to cross-sectionally define the age of onset and the severity of age-associated impairments in acquisition of this relatively difficult hippocampally dependent task. Using a strict behavioral criterion of 80% conditioned responses (CRs), age-associated learning impairments were significant by 24 months of age. Among rabbits that successfully reached this criterion, impairments in acquisition plateaued at 30 months of age. However, the proportion of severely impaired rabbits (that failed to reach the 80% criterion) continued to increase age dependently. Using an easier criterion of 8 out of 10 CRs, behavioral impairments were not detected until 30 months of age, and cases of severe impairment (failure to reach criterion) were rare. Additional controls demonstrated that the deficits observed were not attributable to nonassociative changes that might have artifactually skewed the data. Even severely impaired 36-month-old rabbits were able to reach a criterion of 80% CRs when switched from a trace to a delay conditioning task that is not hippocampally dependent. The results are discussed in terms of operationally defining and predicting behavioral effects of aging, hypothetical neural mechanisms, and efficient experimental design.

Age-related loss of calcium binding proteins in rabbit hippocampus.

Using immunocytochemistry hippocampal levels of the calcium binding proteins calbindin 28K (CB) and parvalbumin (PV) was studied in young (1 month) to very old (60 month) Albino rabbits. Young (3 month) and senescent (30 month) Wistar rats were also examined to compare the distribution and age dependency of PV and CB in both species. The distribution of PV-ir is similar in the rabbit and rat hippocampus. Aging in both species yielded a small loss of PV-ir in axon terminals. The presence of CB-ir interneurons throughout the hippocampus, and the heavy investment of the dentate gyrus (DG) granular cells with CB-ir was also similar in both species. In rabbits, the number of CB-ir interneurons in the CA1, as well as the density of CB-ir in the DG decreased in the first year of life, and did not change between 12-48 months of age. A secondary reduction in the density of CB-ir in the DG was observed at ages beyond 48 months. A similar loss of CB-ir in the DG occurred in the rat. In the CA1, however, the density of CB-ir was similar in young and aged rats. Another remarkable finding was the total absence of CB-ir in CA1 pyramidal neurons of rabbits at any age. Thus, the distribution and age dependency of PV-ir in the hippocampus is similar in both species. The decline of CB-ir in the DG with advancing age is very prominent and may be related to an altered calcium homeostasis in these cells. However, the absence of CB-ir in the CA1 of rabbits makes a causal role for CB in the functional decline of CA1 pyramidal cells during aging unlikely.

Calcium-dependent afterhyperpolarization and learning in young and aging hippocampus.

Hippocampally-dependent trace eyeblink conditioning has been shown to be affected by aging. Aging animals take more trials to acquire the association and are more likely to be unable to learn the task. Hippocampal neurons show decreased post-burst afterhyperpolarizations (AHPs) and less accomodation after conditioning, in a time-dependent fashion which may relate to the role of hippocampus in learning consolidation. CA1 neurons in aging rabbits show increased AHPs and more accomodation, i.e., they are less excitable, and larger calcium action potentials. These age-related changes may underlie the learning deficits in aging rabbits. The lipophylic calcium channel blocker nimodipine reduces the AHP, accomodation and calcium action potential at low concentrations in aging but not young CA1 neurons. Nimodipine also enhances learning rate in a variety of tasks, including eyeblink conditioning, in aging but not young animals and humans. Altered calcium handling by neurons of aging mammals is a striking change, is pharmacologically manipulable, and may be an important factor in altered learning and cognitive abilities in the aging.

A system for quantitative analysis of associative learning. Part 1. Hardware interfaces with cross-species applications.

This paper describes a reliable, durable, and readily calibrated hardware interface system designed to present sensory stimuli at precise time intervals and to transduce and digitize behavioral data in classical conditioning experiments. It has been extensively tested in a 'model'-associative learning task, conditioning of eyeblink or nictitating membrane responses, but is readily adapted to other behavioral paradigms. Each system can run a pair of conditioned experimental or pseudoconditioned control subjects simultaneously, or collect data from a single subject carrying out two tasks simultaneously. The requirements of the system are defined, based around an inexpensive AT-class MS-DOS microcomputer. The interface hardware needed to present auditory tone conditioned stimuli and corneal airpuff-unconditioned stimuli to training subjects are detailed, with timing signals provided by TTL pulses generated at the digital output ports of an analog-to-digital (A/D) converter. An electronic circuit is described that provides stable inputs to the A/D converter, transducing eyeblink responses to voltage signals opto-electronically, without requiring any invasive attachment of the subject to the subject to the measuring device. The 1-piece eyeblink sensor used (selected for ease of alignment and maintenance) is also discussed. Examples of applications for classical conditioning of rabbits, rats, and human subjects are described. A companion paper describes data-acquisition and control software written as a user-friendly interface for this hardware system.

A system for quantitative analysis of associative learning. Part 2. Real-time software for MS-DOS microcomputers.

Microcomputer software was designed and used to control the timing and delivery of sensory stimuli and to acquire and analyze behavioral data during classical conditioning experiments. The software package runs under DOS 4.x through 6.x (earlier versions run under DOS 3.x) on PC AT-compatible microcomputers coupled with appropriate interface hardware (see Thompson et al., 1994). The software controls timed delivery of up to 8 conditional stimuli. It can collect behavioral data from 2 subjects simultaneously performing the same task (e.g., eyeblink responses) or from a single subject performing 2 different tasks (e.g., both eyeblink and conditional discrimination tasks), permitting its use in a number of experimental paradigms. Digital timing signals are adjustable for different stimulus output systems. Behavior is continuously monitored onscreen, ensuring consistent measurement across trials. Real-time performance measures of the presence or absence of conditioned responses allow coordination with external events (e.g., serum sampling, drug delivery, or single-unit recording). Quantitative measures are generated both for each trial and for complete sessions. Records are stored to disk and can be printed or merged for statistical analyses. Data can be archived on standard media, and internal software utilities translate files for export to PC and Macintosh programs. This system and the hardware described in the preceding paper combine ease of use with extremely replicable behavioral measurements across trials, sessions subjects, cohorts, and studies.

Functional aspects of calcium-channel modulation.

Associative learning is accompanied by a number of changes in the brain, many mediated by calcium. We have used eyeblink conditioning, a well-controlled learning task in animals and humans, to elucidate these changes. Our studies have focused on the hippocampus, a temporal lobe structure known to be important for storage of new information during learning in mammalian brain. Hippocampal neurons show an enhanced firing rate during learning correlated with behavioral acquisition; they also show reduction in a calcium-mediated after-hyperpolarization (AHP), a likely mechanism for their enhanced activity. Aging animals and humans exhibit learning deficits; aging hippocampal neurons show increased AHPs and altered calcium buffering, which contribute to the behavioral learning deficits. Intravenous administration of the calcium antagonist nimodipine causes aging rabbits to learn the eyeblink conditioning task as quickly as young controls. Oral nimodipine enhances learning rates in aging rabbits, rats, and monkeys. In each case, the type of learning task analyzed is dependent on hippocampal processing for acquisition and is impaired with aging. Nimodipine also reverses aging-related alterations in open field behavior of both rats and rabbits. We have done a series of physiological studies focused on the possible role of nimodipine in enhancing neuronal activity in the hippocampus of aging rabbits. The purpose of these studies was to determine how nimodipine may be functioning at a cellular level to increase the learning rate. Four major conclusions may be drawn from our data: (a) Nimodipine strongly enhanced the firing rate of single hippocampal pyramidal neurons recorded in vivo in an aging- and concentration-dependent fashion. Other calcium-channel blockers, such as nifedipine and flunarizine, given to control for cerebral blood flow changes, had essentially no effect on the hippocampal firing rate. (b) The slow AHP, mediated by an outward calcium-activated potassium current, was markedly larger in pyramidal neurons in hippocampal slices prepared from aging rabbits. Nimodipine, at concentrations as low as 100 nM, reliably reduced the AHPs of aging pyramidal cells. Aging neurons also showed more spike frequency adaptation, or accommodation, than young neurons. Nimodipine partially blocked accommodation at concentrations as low as 10 nM in aging neurons. (c) The calcium action potential was larger in aging neurons. Nimodipine modulated the calcium action potential in an age- and concentration-dependent fashion; concentrations as low as 100 nM reduced the calcium action potential in aging CA1 neurons without effects on young cells. (d) Nimodipine blocked the high threshold, noninactivating calcium current (L-type calcium current) in acutely dissociated hippocampal pyramidal neurons.(ABSTRACT TRUNCATED AT 400 WORDS)