Our society is rapidly aging, with the number of seniors expected to double by 2050 (US Census: http://www.census.gov/population/www/pop-profile/elderpop.html
). At the same time, the Information Age requires increasing organizational skills to deal with even basic needs such as medical care and paying bills. However, executive and working memory functions decline early in the normal aging process10–13
, beginning in middle age14,15
. Thus, cognitive changes with advancing age may be costly, forcing retirement from demanding careers and jeopardizing the ability to live independently in an increasingly complex society. Aging monkeys provide an ideal model to reveal the neurobiology of normal aging, as they have a highly developed PFC, but are not subject to age-related dementias16
. Thus, one can be certain that cognitive changes are the result of normal aging and not incipient Alzheimer’s Disease. Like humans, monkeys begin to develop deficits in executive function as early as middle age17
. Both aged monkeys18,19
are impaired on working memory tasks that require constant updating of the contents of memory (Supplementary Information
), bringing to mind information from longer-term stores (e.g. where did I leave my car keys this time?), or keeping in mind a recent event (e.g. remembering a new phone number).
In primates, spatial working memory depends upon the highly evolved dorsolateral PFC6
(). Spatial working memory performance () relies on networks of pyramidal neurons that interconnect at dendritic spines (), and excite each other to keep information “in mind”, i.e. generating persistent spiking activity over a delay period in a working memory task6
(). This ability to maintain information that is no longer in the environment is a fundamental process needed for abstract thought and flexible responding6
. Intracellular signaling pathways modulate the physiological strength of these recurrent, excitatory PFC network connections9
. Recent data show that increased cAMP signaling weakens network connectivity by opening potassium channels, while inhibiting cAMP signaling and/or closing these channels strengthens connectivity and cognitive ability9
(). Specifically, cAMP signaling appears to weaken persistent firing and impair working memory by increasing the open state of HCN (Hyperpolarization-activated Cyclic Nucleotide gated) channels that are localized on spines where networks interconnect8
. Recent data suggest that HCN channels may also gate synaptic inputs through interactions with KCNQ channels, whose open state is increased by cAMP activating protein kinase A (PKA)21
. studies suggest that cAMP signaling is disinhibited in the aged PFC22
. Noradrenergic α2A receptor inhibition of cAMP may be reduced from loss of α2A receptors in the aged PFC23
, and decreased excitation of noradrenergic neurons24
Figure 1 Age-related changes in the PFC networks that subserve working memory. a, The region of the DLPFC most needed for spatial working memory and the site of recordings. PS=principal sulcus; AS=arcuate sulcus. b, The oculomotor delayed response (ODR) spatial (more ...)
There have been few electrophysiological recordings from aged PFC neurons due to the demanding nature of this procedure. Recordings from rat orbital PFC found reduced flexibility in aged neurons25
. However, there have been no in vivo
recordings from the aged dorsolateral PFC, even though behavioral data suggest that this region is particularly vulnerable to normal aging. In vitro
recordings from dorsolateral PFC neurons found relatively subtle changes in excitability with advancing age26
, but their consequence to executive function must be observed in a cognitively-engaged circuit. The current study performed the very first physiological characterization of PFC neuronal response during a working memory task in young adult, middle-aged, and aged monkeys.
Monkeys (macaca mulatta
, n=6) were trained to perform a spatial working memory task in which they must remember a spatial location over a brief delay period; the spatial location changes randomly on each trial (). Two animals were young adults (7 and 9 year old males), two were middle-aged (12 and 13 year old males), and two were aged (17 year old male, 21 year old female). Short delays (2.5s) were used in all age groups to ensure similar performance (>85% percent correct) across age groups. Neurons (n=301) were recorded from area 46, the dorsolateral PFC subregion most needed for visuospatial working memory (). Neurons were characterized based on task-related firing as responsive during a) the visuospatial cue period, b) the delay period when the spatial position was being remembered, and/or c) the motor response period. Some neurons fired only during cue presentation (CUE cells, n=28), while most neurons fired during the delay period as well as to the cue and/or response periods (DELAY cells, n=273). Persistent firing during the delay period is of particular interest, as it is required for working memory6
. Many PFC DELAY neurons elevated their activity during the memory of one spatial location (its preferred direction, shown in blue), but not other locations (the “anti-preferred” direction 180° away from the preferred direction is shown in red; ).
The firing of DELAY cells was markedly reduced with advancing age (, ). portrays the differences in firing rates across the population of DELAY neurons in young, middle-aged and aged animals (individual examples of DELAY neurons in young, middle-aged and aged monkeys are in Supplementary Fig. 1
). There was a significant decline in the spontaneous firing rate of DELAY cells, as well as a marked decline in task-related firing. shows a steep decline in the firing rates of DELAY cells across the age span (t-test on age variable in regression analysis, p<10−4
for all epochs), with older animals showing a restricted range of lower firing rates (Supplementary Fig. 2
). This age-related activity decline persisted throughout the 2.5-s delay period (without main effect of epoch (0.5 s) or age × epoch interaction in repeated measures ANOVA, p>0.25). Additional control analyses showed that age-related decline in the firing rate of DELAY cells is not likely due to a sampling bias during the recording experiment (see Supplementary Information
). The age-related decline in firing rate was particularly prominent during the cue and delay periods for the neuron’s preferred direction (); the decline in firing for the anti-preferred direction () or before target onset () was less pronounced. Consequently, the difference in delay-related firing for the neuron’s preferred direction vs. its anti-preferred direction eroded with increasing age (t-test, p<10−5
, for cue period and every 0.5s epoch in the delay period; ), largely due to reduced firing for the neuron’s preferred direction (). This led to a reduction in d′ with advancing age (t-test on age vs. d′ correlation coefficient, p<0.0001), i.e. a reduced ability to distinguish the preferred from anti-preferred directions during the delay period when spatial information was held in working memory (). These results are consistent with studies showing impairment in spatial working memory in aged monkeys at relatively short (e.g. 5s) delays18
, and single-unit data as well as neural circuit modeling suggest that inadequate PFC recurrent network firing underlies the deficits in PFC cognitive function observed in aging monkeys and humans (Supplementary Figs. 3, 4
; Supplementary Information
Figure 2 Age-dependent decline in the spatially-tuned, persistent firing of DLPFC DELAY neurons. a, Marked reduction of DLPFC DELAY activity for the neurons’ preferred direction with advancing age. Activity of individual neurons of each animal was averaged (more ...)
In contrast to DELAY neurons that showed prominent decline in firing with advancing age, there were no age-related changes in firing rates of PFC CUE cells that responded specifically to the spatial cue (). The average firing rate of these neurons for the preferred direction during the cue period was 26.7±4.4 spikes/s in young monkeys (n=12 neurons), and 25.3±3.7 spikes/s in old monkeys (11 neurons; t-test, p>0.8; significant age × cell type interaction in 2-way ANOVA, p<0.05). These data suggest that reductions in memory-related firing rate do not arise from generalized changes with advancing age affecting all neurons, but rather, are especially evident in recurrent circuits that must maintain firing in the absence of “bottom-up” sensory stimulation.
Figure 3 Firing rates of DLPFC CUE cells remain stable in aged monkeys. The average firing rates of CUE cells in young monkeys (left graph; 10 neurons from a 7-y old and 2 neurons from a 9-y old monkey), did not differ from the firing rate in the oldest monkey (more ...)
What changes in the aging brain contribute to reduced firing during the delay period? There are many brain alterations associated with advancing age27
, including decreased PFC gray matter volume28
, focal changes in white matter29
, and dendritic spine loss30
, all of which correlate with cognitive decline. Importantly, spine loss is especially prominent in layer III- the layer where the recurrent excitatory networks reside- and thin-type spines are the most vulnerable in the aged PFC30
. Immunoelectron microscopy indicates that thin spines have the greatest concentration of cAMP-HCN channel signaling proteins, suggesting that disinhibition of cAMP signaling with advancing age may weaken thin spines in particular9
. Thus, we tested whether inhibition of cAMP signaling in the PFC could partially restore the working memory-related firing of aged neurons, or whether reductions in firing were irreversible due to immutable architectural changes in the aged brain. Drugs were applied near the recorded neurons using iontophoresis, whereby a small electrical current was applied to extrude charged molecules from glass pipettes attached to the recording electrode. Only a minute amount of drug was released, sufficient to alter the firing of nearby neurons, without altering behavioral performance.
Agents that inhibit cAMP signaling or block HCN or KCNQ channels restored persistent firing during the delay period of the working memory task (). For example, iontophoresis of the α2A agonist, guanfacine (), or the cAMP-PKA inhibitor, Rp-cAMPS (), significantly increased firing during the delay period on trials when the cue had appeared at the neuron’s preferred direction. In contrast, the PDE4 inhibitor, etazolate- which increases cAMP signaling- further decreased neuronal firing in aged neurons (p<0.001; Supplementary Fig. 5
). We also tested whether blockade of HCN or KCNQ channels could restore firing, given that cAMP-PKA signaling increases the open state of these ion channels. As shown in , a low dose of the HCN channel blocker, ZD7288 significantly enhanced the delay-related firing rates of neurons in aged monkeys. KCNQ channels were also of interest, as in vitro
physiological characterizations of PFC neurons in aged primate have found increases in the slow afterhyperpolarization (sAHP), which is mediated in part by KCNQ channels26
. As shown in , blockade of KCNQ channels with XE991 increased delay-related firing in aged PFC neurons. Thus, agents that reduced cAMP opening of HCN or KCNQ channels all restored firing rates to levels resembling those observed in younger monkeys. These findings are consistent with behavioral data showing that guanfacine and Rp-cAMPS can enhance working memory performance in aged animals when administered systemically (guanfacine) or directly into the rat PFC (guanfacine or Rp-cAMPS)22
(see Supplementary Information
). Based on these data, guanfacine is currently being tested in elderly humans with PFC cognitive deficits (clinicaltrials.gov: NCT00935493).
Figure 4 Iontophoresis of compounds that inhibit cAMP-PKA signaling, or block HCN or KCNQ channel signaling, strengthens delay-related firing in aged PFC DELAY neurons. a–d,. A summary of the results showing a significant increase in population-average (more ...)
In summary, the current study revealed a physiological basis for age-related working memory decline in the primate brain, with reduction of memory-related firing beginning in middle age and worsening with advancing age. This marked change in network physiology may render higher cortical circuits especially vulnerable to neurodegenerative processes such as Alzheimer’s Disease. However, these studies also uncovered more hopeful data showing that restitution of the proper neurochemical environment can partially restore physiological integrity. These data establish that cognitive changes with advancing age are malleable, and that there is potential to restore at least some cognitive abilities in the elderly. Maintaining strong PFC physiology into advanced age will be an important advantage in an increasingly complex, aging society.