Mechanisms underlying cognitive enhancement and reversal of cognitive deficits in nonhuman primates by the ampakine CX717

Abstract

Rationale: Performance of cognitive tasks in nonhuman primates (NHPs) requires specific brain regions to make decisions under different degrees of difficulty or "cognitive load."

Objective: Local cerebral metabolic activity ([18F]FDG PET imaging) in dorsolateral prefrontal cortex (DLPFC), medial temporal lobe (MTL), and dorsal striatum (DStr) is examined in NHPs performing a delayed-match-to-sample (DMS) task with variable degrees of cognitive load.

Materials and methods: Correlations between cognitive load and degree of brain metabolic activity were obtained with respect to the influence of the ampakine CX717 (Cortex Pharmaceuticals), using brain imaging and recordings of neuronal activity in NHPs and measures of intracellular calcium release in rat hippocampal slices.

Results: Activation of DLPFC, MTL, and DStr reflected changes in performance related to cognitive load within the DMS task and were engaged primarily on high load trials. Similar increased activation patterns and improved performance were also observed following administration of CX717. Sleep deprivation in NHPs produced impaired performance and reductions in brain activation which was reversed by CX717. One potential basis for this facilitation of cognition by CX717 was increased firing of task-specific hippocampal cells. Synaptic mechanisms affected by CX717 were examined in rat hippocampal slices which showed that N-methyl-D-aspartic acid-mediated release of intracellular calcium was reduced in slices from sleep-deprived rats and reversed by application of CX717 to the bathing medium.

Conclusions: The findings provide insight into how cognition is enhanced by CX717 in terms of brain, and underlying neural, processes that are activated on high vs. low cognitive load trials.

Fig. 1

Performance by NHPs on DMS task under conditions of high and low cognitive load. a DMS trial-by-trial performance curves show the mean (±SEM n=9 animals) percentage of correct trials sorted by delay (1–30 s, 5 s blocks) and number of images (two to six) presented in the match phase of the task. Dashed boxes indicate delay and no. of image combinations for what are considered low cognitive load trials (blue box: 1–10 s, two to three images) vs. high cognitive load trials (red box: 21–30 s, five to six images). b DMS performance over the entire session (n=3 sessions) consisting of (1) randomly presented high, low, and normal cognitive load trials (mixed sessions) or sessions consisting of only low or high load trials (exclusive sessions) as defined in a (n=5 animals). Performance on low and high load trials within mixed sessions is segregated for comparison with performance in exclusive sessions comprised of only low or high load trials. There was no significant difference in performance as a function of trial type in either mixed or exclusive sessions. Asterisks: *p<0.01, **p<0.001 refer to comparison with overall mixed session performance (All). c PET images compare local CMR_glc in brain regions activated by performance of DMS task vs. sessions in which the animals watched a video and no task was performed. Images shown are differences obtained by subtraction of no task sessions from DMS sessions. Left PET images at the level of the dorsolateral prefrontal cortex (DLPFC), striatum (DStr) and medial temporal lobe (MTL) show increased metabolic activity of the respective brain areas plus activation of sensory motor cortex (SI) while performing the DMS task. Right differences in CMR_glc shown for the same brain regions between mixed trial sessions and sessions comprised exclusively of high cognitive load trials indicate significantly increased activation in DLPFC, MTL, and DStr, but not in SI. Color scale indicates degree of statistical significance as multiples of t statistic for change in CMR_glc from global mean (see the “Materials and methods” section)

Fig. 2

PET images comparing CMR_glc in sessions composed exclusively of high cognitive load trials to sessions composed exclusively of low cognitive load trials. Difference images computed as in Fig. 1c. Left difference between low and high load sessions. The same three brain regions shown in Fig. 1 (DLPFC, DStr, and MTL) plus the thalamus (Thal.) and visual cortex (VISCX.) showed increased activation on high load vs. low load DMS trials (n=8). Right opposite comparison of high load session PET images subtracted from images in low load sessions indicted an area in the VMPFC with greater activation on low load DMS trials. This same region in the frontal cortex is shown in Fig. 1 for mixed trial sessions. Significance of CMR_glc differences between sessions indicated by the color scale (lower right) as multiples of the t statistic as in Fig. 1c

Fig. 3

Effects of the ampakine CX717 on DMS performance. a CMR_glc during normal (mixed trial session) DMS task performance shown in Fig. 1c in which drug vehicle was injected 5 min prior to start of session. b Additional activation of DLPFC and MTL during performance of DMS task following intravenous injection of the ampakine CX717 (0.8 mg/kg). Color scale indicates degree of significance of changes in CX717 vs. vehicle DMS sessions. c Overall performance during CX717 and vehicle mixed trial sessions for four animals in which CMR_glc comparisons are shown in b. Mean percentage of correct (±SEM) overall session trials on successive days when animals were administered either vehicle or 0.5–1.5 mg/kg CX717. **p<0.001, differences in performance relative to vehicle sessions

Fig. 4

The ampakine CX717 reverses changes in brain region CMR_glc produced by sleep deprivation. PET scans at the level of DLPFC (top) and MTL/Thal (bottom) are shown for successive conditions of normal alert mixed trial DMS test sessions (left); different test sessions conducted following 30–32 h sleep deprivation (center); and sleep deprivation test sessions in which CX717 was administered (right). Effects of CX717 (0.8 mg/kg, iv) on CMR_glc in sleep-deprived animals (n=9) as reported in Porrino et al. (2005). Color scales (inset right) in PET scans indicate significant (t statistic) increases (yellow–orange) or decreases (blue) in CMR_glc for difference images of normal vehicle DMS sessions vs. no task condition (left); sleep-deprived DMS sessions vs. normal vehicle DMS sessions (center); and sleep-deprived+CX717 DMS sessions vs. sleep-deprived DMS sessions (right). Arrows indicate suggested neural processing “loops” from MTL to PFC and Thal to PFC disrupted by sleep deprivation. Decreased CMR_glc in DLPFC combined with increased CMR_glc in MTL following sleep deprivation and reversal by CX717 can be contrasted to lack of reversal in the Thal/PFC loop (Porrino et al. 2005)

Fig. 5

Enhancement of neuronal firing in the hippocampus following the administration of CX717. Hippocampal category-selective neuron fires in response to multiple images within a category (Hampson et al. 2004). Left sample images presented in DMS task and response to other similar images (underscored) in the same category (probes). Center neural firing of a category cell during DMS task following vehicle or CX717 (1.0 mg/kg). Upper rastergram and trial-based histogram of firing of a category cell to images with a human face. Each row of dots is the occurrence of action potential reflecting cell firing on a single trial in which an image containing one or more faces was presented. Red dots indicate behavioral responses to the sample and match image presentations (vertical lines). Histogram below raster is the sum of spikes from the same cell across all trials in the session normalized in 250-ms bins to spikes per second (Hz). Lower recordings from same CA1 neuron after CX717 (1.0 mg/kg, iv) was administered during the session showed a significant increase in sample peak firing rate (vehicle, 15.2±0.3 Hz; CX717, 20.3±0.5 Hz; F_(1,1588)=12.4, p<0.001) and prolonged the duration of stimulus elicited discharge. Upper right rastergrams and trial-based histograms of a hippocampal delay cell which fired only during the delay phase (2–17 s on horizontal axis) of the DMS task. Lower right administration of CX717 (1.0 mg/kg I.V.) significantly increased the frequency of sustained firing during the delay phases of the task (vehicle, 9.3±0.2 Hz; CX717, 18.7±0.7 Hz; F_(1,1588)=24.7, p<0.001) as shown in the lower trial-based histogram (CX717). DMS trial events are indicated on the horizontal axis, start ring arrow, sample and match presentation indicated by vertical lines at 0.0 and 17 s, respectively. Juice reinforcement delivered at the time of correct match response. Single neurons in the hippocampus were recorded using tungsten electrodes (tip diameters 2–5 μM) (Hampson et al. 2004)

Fig. 6

Confocal fluorescent imaging of intracellular calcium in slices of rat hippocampus. a and b Changes detected by intracellular calcium green fluorescence (illustrated in b) relative to background fluorescence (ΔF/F) produced by bath application of NMDA (10 μM, black bar on horizontal axis). b Images of fluorescence for multiple CA1 cells in a single slice are shown before and after perfusion with different agents. Colored traces (left) indicate the mean time-dependent increase in fluorescence from multiple slices (n=9 slices; three to eight cells analyzed per slice) corresponding to the increase in intracellular calcium concentration following transient exposure to NMDA (10 μM) in the bathing medium as shown in b (NMDA). Slices were exposed to NMDA alone (green trace), then to a combination of NMDA and one or more other agents known to modulate NMDA receptor function. Coadministration of the ampakine CX717 (20 μM) with NMDA produced an increase in NMDA peak fluorescence (blue trace). Exposure with the AMPA receptor antagonist NBQX (20 μM) blocked the increase in NMDA fluorescence produced by CX717 (red trace). Application with the NMDA receptor antagonist CNQX (20 μM) decreased the NMDA-induced increase (dashed red trace) and also suppressed the increase produced by coadministration of CX717 (dashed purple trace). NMDA was delivered as a transient (100 s) perfusion in the bathing medium, and slices were exposed to normal medium (ACSF) before and after exposure to each agent. Slices were exposed to CX717, NBQX, and CNQX in combination with NMDA for 10 min after transient (6 min) exposure to NMDA alone. c Mean change in peak fluorescence (as shown in a) across all cells in which NMDA was coadministered with CX717, NBQX, and/or CNQX. Asterisks indicate significant differences with respect to NMDA alone (**p<0.001). d Area of 300 μm thick hippocampal slices (extracted from adult rats aged 45–65 days) showing CA1 cell layer in which pyramidal cells were imaged

Fig. 7

Sleep deprivation induced decrease in NMDA-mediated intracellular calcium flux is reversed by CX717. a Mean change in calcium green fluorescence following exposure to NMDA (10 μM) and NMDA+CX717 (20 μM) in slices from alert 45- to 65-day-old rats (n=3 rats, three slices per rat, four to eight cells per slice). Slices were exposed to normal medium (5 min) before and after exposure to NMDA (solid line). Slices were exposed to CX717 (dashed line) in the bathing medium for 10 min prior to as well as during NMDA exposure. b Effects of NMDA and CX717 in slices from sleep-deprived (Sleep Dep.) rats (n=3). Traces indicate mean change in calcium green fluorescence in response to NMDA (solid line) and NMDA+CX717 (dashed line) in slices (n=10 slices, five to nine cells per slice) prepared immediately following 48 h sleep deprivation procedure. c Mean peak fluorescence indicating increased intracellular calcium concentration in response to NMDA (gray bar) and NMDA+CX717 (striped bar) in slices from alert (nonsleep-deprived) rats as described in a. *p<0.010, significant increase NMDA+CX717 vs. NMDA alone. d Mean peak fluorescence for NMDA (gray bar) and NMDA+CX717 (striped bar) in slices from sleep-deprived (Sleep Dep.) rats shown in b. **p<0.001, significant decrease in NMDA elicited peak in slices from sleep-deprived vs. alert rats; ‡p<0.001, significant increase in NMDA+CX717 vs. NMDA alone in sleep-deprived slices. NMDA+CX717 fluorescence was not significantly different in alert vs. sleep-deprived slices