Aging-Related Alterations to Persistent Firing in the Lateral Entorhinal Cortex Contribute to Deficits in Temporal Associative Memory.

With aging comes a myriad of different disorders, and cognitive decline is one of them. Studies have consistently shown a decline amongst aged subjects in their ability to acquire and maintain temporal associative memory. Defined as the memory of the association between two objects that are separated in time, temporal associative memory is dependent on neocortical structures such as the prefrontal cortex and temporal lobe structures. For this memory to be acquired, a mental trace of the first stimulus is necessary to bridge the temporal gap so the two stimuli can be properly associated. Persistent firing, the ability of the neuron to continue to fire action potentials even after the termination of a triggering stimulus, is one mechanism that is posited to support this mental trace. A recent study demonstrated a decline in persistent firing ability in pyramidal neurons of layer III of the lateral entorhinal cortex with aging, contributing to learning impairments in temporal associative memory acquisition. In this work, we explore the potential ways persistent firing in lateral entorhinal cortex (LEC) III supports temporal associative memory, and how aging may disrupt this mechanism within the temporal lobe system, resulting in impairment in this crucial behavior.

Variability in sub-threshold signaling linked to Alzheimer's disease emerges with age and amyloid plaque deposition in mouse ventral CA1 pyramidal neurons.

The hippocampus is vulnerable to deterioration in Alzheimer's disease (AD). It is, however, a heterogeneous structure, which may contribute to the differential volumetric changes along its septotemporal axis during AD progression. Here, we investigated amyloid plaque deposition along the dorsoventral axis in two strains of transgenic AD (ADTg) mouse models. We also used patch-clamp physiology in these mice to probe for functional consequences of AD pathogenesis in ventral hippocampus, which we found bears significantly higher plaque burden in the aged ADTg group compared to corresponding dorsal regions. Despite dorsoventral differences in amyloid load, ventral CA1 pyramidal neurons of aged ADTg mice exhibited subthreshold physiological changes similar to those previously reported in dorsal neurons, indicative of an HCN channelopathy, but lacked exacerbated suprathreshold accommodation. Additionally, HCN channel function could be rescued by pharmacological manipulation of the endoplasmic reticulum. These observations suggest that an AD-linked HCN channelopathy emerges in both dorsal and ventral CA1 pyramidal neurons, but that the former encounter an additional integrative obstacle in the form of reduced intrinsic excitability.

Intact Female Mice Acquire Trace Eyeblink Conditioning Faster than Male and Ovariectomized Female Mice.

Female subjects have been widely excluded from past neuroscience work because of a number of biases, including the notion that cycling sex hormones increase variability. However, it is necessary to conduct behavioral research in mice that includes both sexes as mice are typically used for developing and evaluating future therapeutics. Understanding sex differences in learning is fundamental for the development of targeted therapies for numerous neurologic and neurodegenerative disorders, including Alzheimer's disease, which is more prevalent in females than males. This study set out to confirm the role of sex and necessity of circulating ovarian hormones in the acquisition of the temporal associative memory task trace eyeblink conditioning (tEBC) in C57BL/6J mice. We present evidence that sex and ovarian hormones are important factors in learning. Specifically, intact female mice learn significantly faster than both male and ovariectomized (ovx) female mice. Data from pseudoconditioned control mice indicate that sex differences are because of differences in learned associations, not sensitization or spontaneous blink rate. This study strengthens the idea that ovarian hormones such as estrogen and progesterone significantly influence learning and memory and that further research is needed to determine the underlying mechanisms behind their effects. Overall, our findings emphasize the necessity of including both sexes in future behavioral studies.

Cognitive aging is associated with redistribution of synaptic weights in the hippocampus.

Behaviors that rely on the hippocampus are particularly susceptible to chronological aging, with many aged animals (including humans) maintaining cognition at a young adult-like level, but many others the same age showing marked impairments. It is unclear whether the ability to maintain cognition over time is attributable to brain maintenance, sufficient cognitive reserve, compensatory changes in network function, or some combination thereof. While network dysfunction within the hippocampal circuit of aged, learning-impaired animals is well-documented, its neurobiological substrates remain elusive. Here we show that the synaptic architecture of hippocampal regions CA1 and CA3 is maintained in a young adult-like state in aged rats that performed comparably to their young adult counterparts in both trace eyeblink conditioning and Morris water maze learning. In contrast, among learning-impaired, but equally aged rats, we found that a redistribution of synaptic weights amplifies the influence of autoassociational connections among CA3 pyramidal neurons, yet reduces the synaptic input onto these same neurons from the dentate gyrus. Notably, synapses within hippocampal region CA1 showed no group differences regardless of cognitive ability. Taking the data together, we find the imbalanced synaptic weights within hippocampal CA3 provide a substrate that can explain the abnormal firing characteristics of both CA3 and CA1 pyramidal neurons in aged, learning-impaired rats. Furthermore, our work provides some clarity with regard to how some animals cognitively age successfully, while others' lifespans outlast their "mindspans."

Persistent firing in LEC III neurons is differentially modulated by learning and aging.

Whether and how persistent firing in lateral entorhinal cortex layer III (LEC III) supports temporal associative learning is still unknown. In this study, persistent firing was evoked in vitro from LEC III neurons from young and aged rats that were behaviorally naive or trained on trace eyeblink conditioning. Persistent firing ability from neurons from behaviorally naive aged rats was lower compared to neurons from young rats. Neurons from learning impaired aged animals also exhibited reduced persistent firing capacity, which may contribute to aging-related learning impairments. Successful acquisition of the trace eyeblink task, however, increased persistent firing ability in both young and aged rats. These changes in persistent firing ability are due to changes to the afterdepolarization, which may in turn be modulated by the postburst afterhyperpolarization. Together, these data indicate that successful learning increases persistent firing ability and decreases in persistent firing ability contribute to learning impairments in aging.

Learning and aging affect neuronal excitability and learning.

The first study that demonstrated a change in intrinsic neuronal excitability after learning in ex vivo brain tissue slices from a mammal was published over thirty years ago. Numerous other manuscripts describing similar learning-related changes have followed over the years since the original paper demonstrating the postburst afterhyperpolarization (AHP) reduction in CA1 pyramidal neurons from rabbits that learned delay eyeblink conditioning was published. In addition to the learning-related changes, aging-related enlargement of the postburst AHP in CA1 pyramidal neurons have been reported. Extensive work has been done relating slow afterhyperpolarization enhancement in CA1 hippocampus to slowed learning in some aging animals. These reproducible findings strongly implicate modulation of the postburst AHP as an essential cellular mechanism necessary for successful learning, at least in learning tasks that engage CA1 hippocampal pyramidal neurons.

CREB overexpression in dorsal CA1 ameliorates long-term memory deficits in aged rats.

The molecular mechanisms underlying age-related cognitive deficits are not yet fully elucidated. In aged animals, a decrease in the intrinsic excitability of CA1 pyramidal neurons is believed to contribute to age-related cognitive impairments. Increasing activity of the transcription factor cAMP response element-binding protein (CREB) in young adult rodents facilitates cognition, and increases intrinsic excitability. However, it has yet to be tested if increasing CREB expression also ameliorates age-related behavioral and biophysical deficits. To test this hypothesis, we virally overexpressed CREB in CA1 of dorsal hippocampus. Rats received CREB or control virus, before undergoing water maze training. CREB overexpression in aged animals ameliorated the long-term memory deficits observed in control animals. Concurrently, cells overexpressing CREB in aged animals had reduced post-burst afterhyperpolarizations, indicative of increased intrinsic excitability. These results identify CREB modulation as a potential therapy to treat age-related cognitive decline.

CREB, cellular excitability, and cognition: Implications for aging.

Humans and laboratory animals display cognitive deficits as they age. However, there are currently no effective therapies available to treat these deficits, as the underlying mechanisms are poorly understood. Studies using pharmacological compounds have found a link between cognitive performance and the intrinsic cellular excitability of CA1 hippocampal neurons. Therefore, it is of great interest to identify molecular regulators that may be influencing both cognition and neuronal excitability, which could be changed with age. One possible regulator is the transcription factor cAMP response element binding-protein (CREB). In young adult animals, manipulation of CREB activity has resulted in modulation of both cognitive performance on behavioral tasks, and neuronal excitability. While evidence is sparse, studies also point to a dysfunction in CREB signaling with aging. We propose that CREB may be a viable therapeutic target for the treatment of age-related cognitive deficits, along with potential experiments to test this hypothesis.

Intrinsic Hippocampal Excitability Changes of Opposite Signs and Different Origins in CA1 and CA3 Pyramidal Neurons Underlie Aging-Related Cognitive Deficits.

Aging-related cognitive deficits have been attributed to dysfunction of neurons due to failures at synaptic or intrinsic loci, or both. Given the importance of the hippocampus for successful encoding of memory and that the main output of the hippocampus is via the CA1 pyramidal neurons, much of the research has been focused on identifying the aging-related changes of these CA1 pyramidal neurons. We and others have discovered that the postburst afterhyperpolarization (AHP) following a train of action potentials is greatly enlarged in CA1 pyramidal neurons of aged animals. This enlarged postburst AHP is a significant factor in reducing the intrinsic excitability of these neurons, and thus limiting their activity in the neural network during learning. Based on these data, it has largely been thought that aging-related cognitive deficits are attributable to reduced activity of pyramidal neurons. However, recent in vivo and ex vivo studies provide compelling evidence that aging-related deficits could also be due to a converse change in CA3 pyramidal neurons, which show increased activity with aging. In this review, we will incorporate these recent findings and posit that an interdependent dynamic dysfunctional change occurs within the hippocampal network, largely due to altered intrinsic excitability in CA1 and CA3 hippocampal pyramidal neurons, which ultimately leads to the aging-related cognitive deficits.

Aging-Related Hyperexcitability in CA3 Pyramidal Neurons Is Mediated by Enhanced A-Type K+ Channel Function and Expression.

Aging-related impairments in hippocampus-dependent cognition have been attributed to maladaptive changes in the functional properties of pyramidal neurons within the hippocampal subregions. Much evidence has come from work on CA1 pyramidal neurons, with CA3 pyramidal neurons receiving comparatively less attention despite its age-related hyperactivation being postulated to interfere with spatial processing in the hippocampal circuit. Here, we use whole-cell current-clamp to demonstrate that aged rat (29-32 months) CA3 pyramidal neurons fire significantly more action potentials (APs) during theta-burst frequency stimulation and that this is associated with faster AP repolarization (i.e., narrower AP half-widths and enlarged fast afterhyperpolarization). Using a combination of patch-clamp physiology, pharmacology, Western blot analyses, immunohistochemistry, and array tomography, we demonstrate that these faster AP kinetics are mediated by enhanced function and expression of Kv4.2/Kv4.3 A-type K(+) channels, particularly within the perisomatic compartment, of CA3 pyramidal neurons. Thus, our study indicates that inhibition of these A-type K(+) channels can restore the intrinsic excitability properties of aged CA3 pyramidal neurons to a young-like state. Significance statement: Age-related learning deficits have been attributed, in part, to altered hippocampal pyramidal neuronal function with normal aging. Much evidence has come from work on CA1 neurons, with CA3 neurons receiving comparatively less attention despite its age-related hyperactivation being postulated to interfere with spatial processing. Hence, we conducted a series of experiments to identify the cellular mechanisms that underlie the hyperexcitability reported in the CA3 region. Contrary to CA1 neurons, we demonstrate that postburst afterhyperpolarization is not altered with aging and that aged CA3 pyramidal neurons are able to fire significantly more action potentials and that this is associated with faster action potential repolarization through enhanced expression of Kv4.2/Kv4.3 A-type K(+) channels, particularly within the cell bodies of CA3 pyramidal neurons.

Increased Excitability of Both Principal Neurons and Interneurons during Associative Learning.

In this review, we highlight several studies indicating that the modulation of intrinsic neuronal excitability is key for successful memory formation. Specifically, we will focus our discussion on our hypothesis that the postburst afterhyperpolarization (a key regulator of intrinsic excitability) is an essential cellular mechanism used by both principal and inhibitory neurons to change their neuronal activity as memory is formed. In addition, we propose that these intrinsic excitability changes occur first in principal neurons, followed by changes in inhibitory neurons, thus maintaining the balance of network activity among neurons for successful encoding and readout of memory.

Altered calcium metabolism in aging CA1 hippocampal pyramidal neurons.

Altered neuronal calcium homeostasis is widely hypothesized to underlie cognitive deficits in normal aging subjects, but the mechanisms that underlie this change are unknown, possibly due to a paucity of direct measurements from aging neurons. Using CCD and two-photon calcium imaging techniques on CA1 pyramidal neurons from young and aged rats, we show that calcium influx across the plasma membrane increases with aging, and that this change is countered by increased intracellular calcium buffering. The additional buffer in aging neurons balances the increased calcium influx following a small number (<3) action potentials, but is overwhelmed during sustained or theta-like activity which leads to a greater rise in intracellular calcium concentration in aging than that in young neurons. Our results demonstrate that calcium overload occurs regularly in aging CA1 pyramidal neurons under physiological conditions. This overload may be a critical factor in age-related decline in hippocampus-dependent cognitive function.

Learning increases intrinsic excitability of hippocampal interneurons.

Learning-related intrinsic excitability changes of pyramidal neurons via modulation of the postburst afterhyperpolarization (AHP) have been repeatedly demonstrated in multiple brain regions (especially the hippocampus), after a variety of learning tasks, and in multiple species. While exciting and important, the changes in pyramidal neurons are only a part of the neural circuitry involved in successful learning. For a more complete picture of the dynamic learning-related changes in the neural network, changes in inhibitory circuitry must also be systematically examined and characterized. Here we show in young adult rats and mice that learning the hippocampus-dependent trace eyeblink conditioning task induces enhanced inhibition onto CA1 pyramidal neurons mediated, in part, by an increase in intrinsic excitability of somatostatin-positive inhibitory neurons (SOMs). Furthermore, both CA1 pyramidal and SOM interneurons shared a common cellular mechanism (reduction in SK channel-mediated AHP) that led to the learning-induced increased intrinsic excitability.

Increasing SK2 channel activity impairs associative learning.

Enhanced intrinsic neuronal excitability of hippocampal pyramidal neurons via reductions in the postburst afterhyperpolarization (AHP) has been hypothesized to be a biomarker of successful learning. This is supported by considerable evidence that pharmacologic enhancement of neuronal excitability facilitates learning. However, it has yet to be demonstrated that pharmacologic reduction of neuronal excitability restricted to the hippocampus can retard acquisition of a hippocampus-dependent task. Thus, the present study was designed to address this latter point using a small conductance potassium (SK) channel activator NS309 focally applied to the dorsal hippocampus. SK channels are important contributors to intrinsic excitability, as measured by the medium postburst AHP. NS309 increased the medium AHP and reduced excitatory postsynaptic potential width of CA1 neurons in vitro. In vivo, NS309 reduced the spontaneous firing rate of CA1 pyramidal neurons and impaired trace eyeblink conditioning in rats. Conversely, trace eyeblink conditioning reduced levels of SK2 channel mRNA and protein in the hippocampus. Therefore, the present findings indicate that modulation of SK channels is an important cellular mechanism for associative learning and further support postburst AHP reductions in hippocampal pyramidal neurons as a biomarker of successful learning.

Learning and aging related changes in intrinsic neuronal excitability.

A goal of many laboratories that study aging is to find a key cellular change(s) that can be manipulated and restored to a young-like state, and thus, reverse the age-related cognitive deficits. We have chosen to focus our efforts on the alteration of intrinsic excitability (as reflected by the postburst afterhyperpolarization, AHP) during the learning process in hippocampal pyramidal neurons. We have consistently found that the postburst AHP is significantly reduced in hippocampal pyramidal neurons from young adults that have successfully learned a hippocampus-dependent task. In the context of aging, the baseline intrinsic excitability of hippocampal neurons is decreased and therefore cognitive learning is impaired. In aging animals that are able to learn, neuron changes in excitability similar to those seen in young neurons during learning occur. Our challenge, then, is to understand how and why excitability changes occur in neurons from aging brains and cause age-associated learning impairments. After understanding the changes, we should be able to formulate strategies for reversing them, thus making old neurons function more as they did when they were young. Such a reversal should rescue the age-related cognitive deficits.

Cellular mechanisms for altered learning in aging.

Getting gray hair is part of the natural progression of aging. People expect it and they can change their hair color, if they choose. People also expect increases in memory lapses and learning difficulties as they get older. However, unlike hair color, there is no magic cure or option to fix learning and memory difficulties, because the cellular mechanisms of learning and aging in all the different types of neurons throughout the brain have yet to be discovered. This review describes our efforts to identify a cellular biomarker in hippocampal pyramidal neurons that has been demonstrated to reliably change with learning and with aging - the postburst afterhyperpolarization. We propose that this biomarker, which plays a critical role in regulating neuronal excitability, can be used as a benchmark for future studies in order to understand and identify the cellular mechanisms of learning and aging in the hippocampus, as well as in other cortical regions.

Learning-related postburst afterhyperpolarization reduction in CA1 pyramidal neurons is mediated by protein kinase A.

Learning-related reductions of the postburst afterhyperpolarization (AHP) in hippocampal pyramidal neurons have been shown ex vivo, after trace eyeblink conditioning. The AHP is also reduced by many neuromodulators, such as norepinephrine, via activation of protein kinases. Trace eyeblink conditioning, like other hippocampus-dependent tasks, relies on protein synthesis for consolidating the learned memory. Protein kinase A (PKA) has been shown to be a key contributor for protein synthesis via the cAMP-response element-binding pathway. Here, we have explored a potential involvement of PKA and protein kinase C (PKC) in maintaining the learning-related postburst AHP reduction observed in CA1 pyramidal neurons. Bath application of isoproterenol (1 muM), a beta-adrenergic agonist that activates PKA, significantly reduced the AHP in CA1 neurons from control animals, but not from rats that learned. This occlusion suggests that PKA activity is involved in maintaining the AHP reduction measured ex vivo after successful learning. In contrast, bath application of the PKC activator, (-) indolactam V (0.2 muM), significantly reduced the AHP in CA1 neurons from both control and trained rats, indicating that PKC activity is not involved in maintaining the AHP reduction at this point after learning.

Alterations in intrinsic neuronal excitability during normal aging.

Normal aging subjects, including humans, have difficulty learning hippocampus-dependent tasks. For example, at least 50% of normal aging rabbits and rats fail to meet a learning criterion in trace eyeblink conditioning. Many factors may contribute to this age-related learning impairment. An important cause is the reduced intrinsic excitability observed in hippocampal pyramidal neurons from normal aging subjects, as reflected by an enlarged postburst afterhyperpolarization (AHP) and an increased spike-frequency adaptation (accommodation). In this review, we will focus on the alterations in the AHP and accommodation during learning and normal aging. We propose that age-related increases in the postburst AHP and accommodation in hippocampal pyramidal neurons play an integral role in the learning impairment observed in normal aging subjects.

Learning, aging and intrinsic neuronal plasticity.

In vitro experiments indicate that intrinsic neuronal excitability, as evidenced by changes in the post-burst afterhyperpolarization (AHP) and spike-frequency accommodation, is altered during learning and normal aging in the brain. Here we review these studies, highlighting two consistent findings: (i) that AHP and accommodation are reduced in pyramidal neurons from animals that have learned a task; and (ii) that AHP and accommodation are enhanced in pyramidal neurons from aging subjects, a cellular change that might contribute to age-related learning impairments. Findings from in vivo single-neuron recording studies complement the in vitro data. From these consistently reproduced findings, we propose that the intrinsic AHP level might determine the degree of synaptic plasticity and learning. Furthermore, it seems that reductions in the AHP must occur before learning if young and aging subjects are to learn a task successfully.

Pharmacological and molecular enhancement of learning in aging and Alzheimer's disease.

When animals learn hippocampus-dependent associative and spatial tasks such as trace eyeblink conditioning and the water maze, CA1 hippocampal neurons become more excitable as a result of reductions in the post-burst, slow afterhyperpolarization. The calcium-activated potassium current that mediates this afterhyperpolarization is activated by the calcium influx that occurs when a series of action potentials fire and serves as a modulator of neuronal firing frequency. As a result, spike frequency accommodation is also reduced after learning. Neuronal calcium buffering processes change and/or voltage-dependent calcium currents increase during aging; leading to enhancements in the slow afterhyperpolarization, increased spike frequency accommodation and age-associated impairments in learning. We describe a series of studies done to characterize this learning-specific enhancement in intrinsic neuronal excitability and its converse in aging brain. We have also combined behavioral pharmacology and biophysics in experiments demonstrating that compounds that increase neuronal excitability in CA1 pyramidal neurons also enhance learning rate of hippocampus-dependent tasks, especially in aging animals. The studies reviewed here include those using nimodipine, an L-type calcium current blocker that tends to cross the blood-brain barrier; metrifonate, a cholinesterase inhibitor; CI1017, a muscarinic cholinergic agonist; and galantamine, a combined cholinesterase inhibitor and nicotinic agonist. Since aging is the chief risk factor for Alzheimer's disease, a disease that targets the hippocampus and associated brain regions and markedly impairs hippocampus-dependent learning, these compounds have potential use as treatments for this disease. Galantamine has been approved by the USDA for this purpose. Finally, we have extended our studies to the TG2576 transgenic mouse model of Alzheimer's disease (AD), that overproduces amyloid precursor protein (APP) and increases levels of toxic beta-amyloid in the brain. Not only do these mice show deficits in hippocampus-dependent learning as they age, but their hippocampal neurons show a reduced capacity to increase their levels of intrinsic excitability with reductions in the slow afterhyperpolarization after application of the muscarinic agonist carbachol. These TG2576 APP overproducing mice were crossed with BACE1 knockout mice, that do not produce beta-amyloid because cleavage of APP by the beta-site APP cleaving enzyme 1 (BACE1) is a critical step in its formation. Not only was hippocampus-dependent learning rescued in the bigenic TG2576-BACE1 mice, but the capacity of hippocampal neurons to show normal enhancements of intrinsic excitability was restored. The series of studies reviewed here support our hypothesis that enhancement in intrinsic excitability by reductions in calcium-activated potassium currents in hippocampal neurons is an important cellular mechanism for hippocampus-dependent learning.