Alexandre A. Miasnikov

Induction of behavioral associative memory by stimulation of the nucleus basalis

The nucleus basalis (NB) has been implicated in memory formation indirectly, by lesions, pharmacological manipulations, and neural correlates of learning. Prior findings imply that engagement of the NB during learning promotes memory storage. We directly tested this NB-memory hypothesis by determining whether stimulation of the NB induces behavioral associative memory. Rats were trained either with paired tone (6 kHz) and NB stimulation or with the two stimuli unpaired. We later determined the specificity of cardiac and respiratory behavioral responses to the training tone and several other acoustic frequencies. Paired subjects exhibited frequency generalization gradients with a peak of 6 kHz for both cardiac and respiratory behavior. Unpaired subjects exhibited no generalization gradient. The development of such specific, associative behavioral responses indicates that tone paired with NB stimulation induced behavioral associative memory. The discovery of memory induction by direct activation of the NB supports the NB-memory hypothesis and provides a potentially powerful way to control and investigate neural mechanisms of memory.

Muscarinic dependence of nucleus basalis induced conditioned receptive field plasticity.

Receptive field (RF) plasticity in primary auditory cortex of adult animals, specifically selective increased response to a tonal conditioned stimulus (CS) relative to other frequencies, can be induced both by behavioral conditioning and by pairing a tone with stimulation of the nucleus basalis (NB). This study determined whether cortical muscarinic receptors are necessary for NB-induced RF plasticity. Single units in layers II–IV were studied in Urethane anesthetized adult rats. The cortex was perfused with saline or saline + atropine sulfate. Conditioning, 30 trials of pairing a tone with NB stimulation, produced a significant CS-specific response increase (n = 8). Local atropine blocked NB-induced RF plasticity, actually resulting in CS-specific response decrease (n = 6). Therefore, NB-induced RF plasticity requires engagement of muscarinic receptors in auditory cortex.

The level of cholinergic nucleus basalis activation controls the specificity of auditory associative memory

Learning involves not only the establishment of memory per se, but also the specific details of its contents. In classical conditioning, the former concerns whether an association was learned while the latter discloses what was learned. The neural bases of associativity have been studied extensively while neural mechanisms of memory specificity have been neglected. Stimulation of the cholinergic nucleus basalis (NBs) paired with a preceding tone induces CS-specific associative memory. As different levels of acetylcholine may be released naturally during different learning situations, we asked whether the level of activation of the cholinergic neuromodulatory system can control the degree of detail that is encoded and retrieved. Adult male rats were tested pre- and post-training for behavioral responses (interruption of ongoing respiration) to tones of various frequencies (1-15 kHz, 70 dB, 2 s). Training consisted of 200 trials/day of tone (8.0 kHz, 70 dB, 2 s) either paired or unpaired with NBs (CS-NBs = 1.8 s) at moderate (65.7+/-9.0 microA, one day) or weak (46.7+/-12.1 microA, three training days) levels of stimulation, under conditions of controlled behavioral state (pre-trial stable respiration rate). Post-training (24 h) responses to tones revealed that moderate activation induced both associative and CS-specific behavioral memory, whereas weak activation produced associative memory lacking frequency specificity. The degree of memory specificity 24 h after training was positively correlated with the magnitude of CS-elicited increase in gamma activity within the EEG during training, but only in the moderate NBs group. Thus, a low level of acetylcholine released by the nucleus basalis during learning is sufficient to induce associativity whereas a higher level of release enables the storage of greater experiential detail. gamma waves, which are thought to reflect the coordinated activity of cortical cells, appear to index the encoding of CS detail. The findings demonstrate that the amount of detail in memory can be directly controlled by neural intervention.

The effects of electrical stimulation of the nucleus basalis on the electroencephalogram, heart rate, and respiration

The nucleus basalis (NB) mediates cortical electroencephalograph (EEG) activation; NB stimulation also modulates cortical responses to sensory stimuli and can induce learning-related receptive field plasticity. However, little is known about the behavioral effects of NB stimulation. This study concerns the effects of NB stimulation on cardiac and respiratory behavior and quantifies its EEG effects in freely moving rats. The EEG exhibited stimulation-induced decreases in theta and alpha power and increases in gamma power. NB stimulation elicited biphasic heart rate changes and disrupted ongoing respiration patterns. Neither EEG nor behavioral effects exhibited habituation or facilitation. These results indicate that the NB may serve not only as a cortical, but also as a behavioral, activation system that is normally engaged during learning.

CS-specific gamma, theta, and alpha EEG activity detected in stimulus generalization following induction of behavioral memory by stimulation of the nucleus basalis

Tone paired with stimulation of the nucleus basalis (NB) induces behavioral memory that is specific to the frequency of the conditioned stimulus (CS), assessed by cardiac and respiration behavior during post-training stimulus generalization testing. This paper focuses on CS-specific spectral and temporal features of conditioned EEG activation. Adult male Sprague-Dawley rats, chronically implanted with a stimulating electrode in the NB and a recording electrode in the ipsilateral auditory cortex, received either tone (6kHz, 70dB, 2s) paired with co-terminating stimulation of the nucleus basalis (0.2s, 100Hz, 80-105 microA, ITI approximately 45s) or unpaired presentation of the stimuli (approximately 200 trials/day for approximately 14 days). CS-specificity was tested 24h post-training by presenting test tones to obtain generalization gradients for the EEG, heart rate, and respiration. Behavioral memory was evident in cardiac and respiratory responses that were maximal to the CS frequency of 6kHz. FFT analyses of tone-elicited changes of power in the delta, theta, alpha, beta1, beta2, and gamma bands in the paired group revealed that conditioned EEG activation (shift from lower to higher frequencies) was differentially spectrally and temporally specific: theta, and alpha to a lesser extent, decreased selectively to 6kHz during and for several seconds following tone presentation while gamma power increased transiently during and after 6kHz. Delta exhibited no CS-specificity and the beta bands showed transient specificity only after several seconds. The unpaired group exhibited neither CS-specific behavioral nor EEG effects. Thus, stimulus generalization tests reveal that conditioned EEG activation is not unitary but rather reflects CS-specificity, with band-selective markers for specific, associative neural processes in learning and memory.

Association learning-dependent increases in acetylcholine release in the rat auditory cortex during auditory classical conditioning

The cholinergic system has been implicated in sensory cortical plasticity, learning and memory. This experiment determined the relationship between the acquisition of a Pavlovian conditioned approach response (CR) to an auditory conditioned stimulus (CS) and the release of acetylcholine (ACh) in the primary auditory cortex in rats. Samples of ACh were collected via microdialysis during behavioral training in either an auditory classical conditioning task or in a non-associative control task. The conditioning group received daily pairings of a white noise CS with a sucrose pellet unconditioned stimulus (US), while the control group received an equal number of CS and US presentations, but with these stimuli being presented randomly. Training was conducted on three consecutive days, with microdialysis samples being collected on Days 1 and 3 in separate sub-groups. The level of ACh released in the auditory cortex during conditioning trials increased from the first to the third day of training in the conditioning group as rats acquired the CR, but did not change in the control group, which did not acquire a CR. These data provide direct evidence for the hypothesis that ACh release increases in the primary auditory cortex during natural memory formation, where cholinergic activation is known to contribute to the formation of specific associative representational plasticity in conjunction with specific memory formation.

Sensory memory consolidation observed: Increased specificity of detail over days

Memories are usually multidimensional, including contents such as sensory details, motivational state and emotional overtones. Memory contents generally change over time, most often reported as a loss in the specificity of detail. To study the temporal changes in the sensory contents of associative memory without motivational and emotional contents, we induced memory for acoustic frequency by pairing a tone with stimulation of the cholinergic nucleus basalis. Adult male rats were first tested for behavioral responses (disruption of ongoing respiration) to tones (1-15 kHz), yielding pre-training behavioral frequency generalization gradients (BFGG). They next received three days of training consisting of a conditioned stimulus (CS) tone (8.00 kHz, 70 dB, 2 s) either Paired (n=5) or Unpaired (n=5) with weak electrical stimulation (approximately 48 microA) of the nucleus basalis (100 Hz, 0.2 s, co-terminating with CS offset). Testing for behavioral memory was performed by obtaining post-training BFGGs at two intervals, 24 and 96 h after training. At 24 h post-training, the Paired group exhibited associative behavioral memory manifested by significantly larger responses to tone than the Unpaired group. However, they exhibited no specificity in memory for the frequency of the tonal CS, as indexed by a flat BFGG. In contrast, after 96 h post-training the Paired group did exhibit specificity of memory as revealed by tuned BFGGs with a peak at the CS-band of frequencies. This increased detail of memory developed due to a loss of response to lower and higher frequency side-bands, without any change in the absolute magnitude of response to CS-band frequencies. These findings indicate that the sensory contents of associative memory can be revealed to become more specific, through temporal consolidation in the absence of non-sensory factors such as motivation and emotion.

Consolidation and long-term retention of an implanted behavioral memory

Hypothesized circuitry enabling information storage can be tested by attempting to implant memory directly in the brain in the absence of normal experience. Previously, we found that tone paired with activation of the cholinergic nucleus basalis (NB) does induce behavioral memory that shares cardinal features with natural memory; it is associative, highly specific, rapidly formed, consolidates and shows intermediate retention. Here we determine if implanted memory also exhibits long-term consolidation and retention. Adult male rats were first tested for behavioral responses (disruption of ongoing respiration) to tones (1-15 kHz), yielding pre-training behavioral frequency generalization gradients. They next received 3 days of training with a conditioned stimulus (CS) tone (8.0 kHz, 70 dB, 2s) either paired (n=7) or unpaired (n=6) with moderate electrical stimulation of the nucleus basalis (∼ 65 μA, 100 Hz, 0.2s, co-terminating with CS offset). Testing for long-term retention was performed by obtaining post-training behavioral frequency generalization gradients 24h and 2 weeks after training. At 24h post-training, the Paired group exhibited specific associative behavioral memory, manifested by larger responses to the CS frequency band than the Unpaired group. This memory was retained 2 weeks post-training. Moreover, 2 weeks later, the specificity and magnitude of memory had become greater, indicating that the implanted memory had undergone consolidation. Overall, the results demonstrate the validity of NB-implanted memory for understanding natural memory and that activation of the cholinergic nucleus basalis is sufficient to form natural associative memory.

Gamma band plasticity in sensory cortex is a signature of the strongest memory rather than memory of the training stimulus

Gamma oscillations (∼30-120Hz) are considered to be a reflection of coordinated neuronal activity, linked to processes underlying synaptic integration and plasticity. Increases in gamma power within the cerebral cortex have been found during many cognitive processes such as attention, learning, memory and problem solving in both humans and animals. However, the specificity of gamma to the detailed contents of memory remains largely unknown. We investigated the relationship between learning-induced increased gamma power in the primary auditory cortex (A1) and the strength of memory for acoustic frequency. Adult male rats (n=16) received three days (200 trials each) of pairing a tone (3.66 kHz) with stimulation of the nucleus basalis, which implanted a memory for acoustic frequency as assessed by associatively-induced disruption of ongoing behavior, viz., respiration. Post-training frequency generalization gradients (FGGs) revealed peaks at non-CS frequencies in 11/16 cases, likely reflecting normal variation in pre-training acoustic experiences. A stronger relationship was found between increased gamma power and the frequency with the strongest memory (peak of the difference between individual post- and pre-training FGGs) vs. behavioral responses to the CS training frequency. No such relationship was found for the theta/alpha band (4-15 Hz). These findings indicate that the strength of specific increased neuronal synchronization within primary sensory cortical fields can determine the specific contents of memory.

Behavioral memory induced by stimulation of the nucleus basalis: Effects of contingency reversal

Specific behavioral associative memory induced by stimulation of the cortically-projecting cholinergic nucleus basalis (NB) is dependent on intrinsic acetylcholine and shares with natural memory such features as associativity, specificity, rapid formation, consolidation and long-term retention. Herein, we examined extinction and the effects of stimulus pre-exposure. Two groups of adult male rats (n=4 each) were first tested for behavioral responses (disruption of ongoing respiration) to tones (1-15 kHz), constituting a pre-training behavioral frequency generalization gradient (BFGG). They next received a first session of training, 200 trials of a tone (8.00 kHz, 70 dB, 2 s) either paired with electrical stimulation of the NB (100 Hz, 0.2 s, approximately 67 microA, NBstm) (group IP) or unpaired (group IU). Twenty-four hours later, they were tested for behavioral memory by obtaining post-training BFGGs. Then the contingencies were reversed yet another 24 h later; the IP group received tone and NBstm unpaired and the IU group received them paired. A final set of generalization gradients was obtained the next day. All stimuli were presented with subjects under state control indexed by regular respiration. Tested 24 h post-initial training, the IP group developed specific associative behavioral memory indicated by increased responses only to CS-band frequencies, while the IU group did not. After subsequent training with unpaired stimuli, the IP group exhibited experimental extinction. Furthermore, after initial exposure to the CS and NBstm unpaired, the IU group exhibited a tendency toward reduced conditioning to CS/NBstm pairing and a significant increase in latency of conditioned responses. The present findings provide additional support for the hypothesis that engagement of the NB is sufficient to induce natural associative memory and suggest that activation of the NB may be a normal component in the formation of natural associative memory.