Motivationally neutral stimulation of the nucleus basalis induces specific behavioral memory

Abstract

The cholinergic system has been implicated in learning and memory. The nucleus basalis (NB) provides acetylcholine (ACh) to the cerebral cortex. Pairing a tone with NB stimulation (NBstm) to alter cortical state induces both associative specific tuning plasticity in the primary auditory cortex (A1) and associative specific auditory behavioral memory. NB-induced memory has major features of natural memory that is induced by pairing a tone with motivational reinforcers, e.g., food or shock, suggesting that the cholinergic system may be a "final common pathway" whose activation promotes memory storage. Alternatively, NB stimulation might itself be motivationally significant, either rewarding or punishing. To investigate these alternatives, adult male rats (n=7) first formed a specific NB-induced memory (CS=8.0kHz, 2.0s paired with NBstm, ISI=1.8s, 200 trials), validated by post-training (24h) frequency generalization gradients (1-15kHz) of respiration interruption that were specific to the CS frequency. Thereafter, they received the same level of NBstm that had induced memory, while confined to one quadrant of an arena, and later tested for place-preference, i.e., avoidance or seeking of the quadrant of NBstm. This NBstm group exhibited neither preference for nor against the stimulated quadrant, compared to sham-operated subjects (n=7). The findings indicate that specific associative memory can be induced by direct activation of the NB without detectable motivational effects of NB stimulation. These results are concordant with a memory-promoting role for the nucleus basalis that places it "downstream" of motivational systems, which activate it to initiate the storage of the current state of its cholinergic targets.

Figure 1

Respiration signal and its quantification. (A) An example of a regular sinusoidal baseline respiration record disrupted by tone presentation. (B) Quantification of the respiration record shown in (A). The “Respiration Change Index” (RCI, see Methods) is sensitive to both increases and decreases in signal amplitude and frequency.

Figure 2

Changes in the auditory cortical EEG during tone-NBstm pairing, and NB stimulation sites. (A) Example of changes in the EEG frequency bands that exhibited the largest changes in power. “All Bands”: original records obtained with band-pass filters set at 1–1000 Hz. “Alpha” and “Gamma”: corresponding records band-passed with digital filters set at 8.8–14.6 Hz to emphasize alpha and 33–59 Hz to emphasize gamma bands, respectively. This example is for a pairing trial (tone = 8.0 kHz, 70 db, 2s, with 200 ms overlapping train, 100 Hz, 60 µA bipolar stimulation of the NB) in unanesthetized rat. Black bars show tone and NBstim. Note the EEG activation, including a distinct decrease in higher voltage, slower waves (“Alpha”) and increase in lower voltage faster waves (“Gamma”). During pairing, EEG activation reflects a combination of effects of the NBstm and its preceding tone. (B) Group mean EEG spectral changes relative to pre-NBstm, computed as the EEG Power Change Index: EEG PCI_i = (Post_i − Pre) / (Post_i + Pre) where the “Pre” period was the mean of the first 2 s out of four immediately preceding tone onset and post measures were calculated for consecutive periods of 1 s. Note major effects are an increase in gamma (closed triangles) and a decrease in alpha (closed circles) power. (C) Stimulating loci reconstructed for the NB-Mem animals. Sites of nucleus basalis stimulation projected onto outlines of frontal section at closest relevant sections anterior to posterior (AP) distance caudal from Bregma in millimeters (Paxinos & Watson, 1997). In all animals, stimulation was within the caudal nucleus basalis (ventrolateral internal capsule, ventromedial lateral globus pallidus and nucleus basalis of Meynart) which projects preferentially to the auditory cortex. The stimulation sites in 6 animals were found at AP −2.3, and in one animal — at AP −1.8 (insert). Abbreviations: B, basal nucleus of Meynert; CeM, amygdala central nucleus medial; CeL, amygdala central nucleus lateral; CPu, caudate–putamen; IC, internal capsule; IPAC, interstitial nucleus of posterior limb of anterior commissure; LGP, lateral globus pallidus; LH, lateral hypothalamus; SI, substantia innominata; SIB, substantia innominata, basal; SIV, substantia innominata, ventral.

Figure 3

Effectiveness of NBstm and tone alone on the auditory cortex after completion of the place-preference test, and comparison with EEG activation during tone-NBstm pairing. Subjects received 200 presentations each of tone and NBstm randomly. (A) Example of EEG activation by NBstm (200 ms, 100 Hz, 60 µA bipolar stimulation) observed for “All Bands”, “Alpha” and “Gamma” bands, as described in Figure 2. As for EEG activation during tone-NBstm pairing, there is a shift from higher voltage, slower waves to lower voltage, faster waves (“All Bands”) with pronounced decrease in alpha activity and increase in gamma activity. Thick narrow bar indicates 200 ms period of NBstm. (B) Group mean spectral changes in EEG induced by NBstm alone. NB stimulation-induced spectral changes in the EEG relative to pre-stimulation period (first 2 s out of 4 s immediately preceding NBstm, computed as in Figure 2. (C) Comparison of EEG activation during training and after place-preference test. The graph shows the mean “EEG Activation Index” (EAI) for tone + NBstim during pairing, NBstm alone and also tone alone after place-preference test. Note that there was no significant difference between the peak magnitudes of the EAI values (p > 0.15 for designated data points), indicating that NBstm potency had not diminished after induction of specific memory during training. During tone-NBstm pairing, both stimuli contribute to the magnitude of EEG activation. However, the contributions of the tone preceding presentation of the NBstm can be observed in the first two data points following tone onset. This probably reflects conditioned EEG activation (McLin, Miasnikov and Weinberger, 2003). Tone alone after the place-preference test still elicits considerable EEG activation although obtained in the absence of pairing (i.e, potential extinction period). Its peak EAI is not significantly different from activation to tone during pairing with NBstm (p > 0.10), suggesting conditioned EEG activation is robust. (D) EEG spectral changes to tone alone after place-preference test. Although smaller in magnitude, note the similarity of the EEG effects of tone alone with the tone-NBstm pairing (Figure 2B) and NBstm alone (Figure 3B).

Figure 4

Respiration frequency generalization gradients. (A) Sample respiratory waveforms obtained from an NB-Mem subject. Shown are responses to three frequencies, the CS (8.00 kHz) and a lower (2.75 kHz) and higher (15.00 kHz) frequencies on the pre-training day (Before) and 24 h post-training (After). RCI values are the quantified values for each record. Note the large disruption of respiration only at the CS frequency after training (RCI = 0.34). The thick horizontal bars indicate tone presentation. (B) CS-specific induced memory as indexed by differential responses to tone after pairing with NBstm. Group mean respiration responses (RCI) (mean ± SE) for all test tone frequencies (X-axis, square frame for 8 kHz denotes CS frequency during training) for the NB-Mem group. Post-training generalization gradients (“After”, black bars) exhibited CS-specific changes for the group (bracket combining responses at 6.25–11.50 kHz; p < 0.001). The individual frequencies, the CS (8.0 kHz, p < 0.05) and the nearby frequency of 11.5 kHz (p < 0.02) each elicited significantly larger responses following pairing. In the figure, statistically significant paired comparisons (post hoc Tukey’s tests) are indicated with asterisks: *p < 0.05 and ***p < 0.005.

Figure 5

Place Preference data obtained from animals in NB-Mem and Cont groups. (A) Schematic representation of the arena (Methods). (B) Relative baseline time spent and number of visits for each quadrant (percent of time spent and number of visits for each quadrant). There was no quadrant bias (see Results). (C) The quadrants were ranked based both on the relative time each particular subject spent in them and the relative number of visits during the baseline (Day 1) expressed as percent of total time and visits to all quadrants. The ranking were the same for time spent and number of visits. Preferences were idiosyncratic to each rat, but the NB-Mem and Cont groups did not differ in the relative amount of time spent in or number of visits to the most to least attended quadrant (see Results). The quadrant where an animal spent the second least relative (in percent) time was selected (arrow) for confinement + NBstm (NB-Mem group) or only confinement (Cont group). (D) Group behavior for the confinement quadrant. Shown are the percent differences in time spent and number of visits between the groups (Cont minus NB-Mem) before (Day 1) and following (Day 3) treatment. The denominators for each measure were the total time spent in and the total number of visits to all four quadrants (same as shown on Y-axes in Fig. 5B and C). See also Table 1. Analysis of treatment effects was accomplished by 2 × 2 repeated measures ANOVA on absolute values (see text). There were no significant effects.