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."

In Vivo Multi-Day Calcium Imaging of CA1 Hippocampus in Freely Moving Rats Reveals a High Preponderance of Place Cells with Consistent Place Fields.

Calcium imaging using GCaMP indicators and miniature microscopes has been used to image cellular populations during long timescales and in different task phases, as well as to determine neuronal circuit topology and organization. Because the hippocampus (HPC) is essential for tasks of memory, spatial navigation, and learning, calcium imaging of large populations of HPC neurons can provide new insight on cell changes over time during these tasks. All reported HPC in vivo calcium imaging experiments have been done in mouse. However, rats have many behavioral and physiological experimental advantages over mice. In this paper, we present the first (to our knowledge) in vivo calcium imaging from CA1 hippocampus in freely moving male rats. Using the UCLA Miniscope, we demonstrate that, in rat, hundreds of cells can be visualized and held across weeks. We show that calcium events in these cells are highly correlated with periods of movement, with few calcium events occurring during periods without movement. We additionally show that an extremely large percent of cells recorded during a navigational task are place cells (77.3±5.0%, surpassing the percent seen during mouse calcium imaging), and that these cells enable accurate decoding of animal position and can be held over days with consistent place fields in a consistent spatial map. A detailed protocol is included, and implications of these advancements on in vivo imaging and place field literature are discussed.Significance statement:In vivo calcium imaging in freely moving animals allows the visualization of cellular activity across days. In this paper, we present the first in vivo Ca2+ recording from CA1 hippocampus in freely moving rats. We demonstrate that hundreds of cells can be visualized and held across weeks, and that calcium activity corresponds to periods of movement. We show that a high percentage (77.3±5.0%) of imaged cells are place cells, and that these place cells enable accurate decoding and can be held stably over days with little change in field location. Because the hippocampus is essential for many tasks involving memory, navigation, and learning, imaging of large populations of HPC neurons can shed new insight on cellular activity changes and organization.

Place cells are nonrandomly clustered by field location in CA1 hippocampus.

A challenge in both modern and historic neuroscience has been achieving an understanding of neuron circuits, and determining the computational and organizational principles that underlie these circuits. Deeper understanding of the organization of brain circuits and cell types, including in the hippocampus, is required for advances in behavioral and cognitive neuroscience, as well as for understanding principles governing brain development and evolution. In this manuscript, we pioneer a new method to analyze the spatial clustering of active neurons in the hippocampus. We use calcium imaging and a rewarded navigation task to record from 100 s of place cells in the CA1 of freely moving rats. We then use statistical techniques developed for and in widespread use in geographic mapping studies, global Moran's I, and local Moran's I to demonstrate that cells that code for similar spatial locations tend to form small spatial clusters. We present evidence that this clustering is not the result of artifacts from calcium imaging, and show that these clusters are primarily formed by cells that have place fields around previously rewarded locations. We go on to show that, although cells with similar place fields tend to form clusters, there is no obvious topographic mapping of environmental location onto the hippocampus, such as seen in the visual cortex. Insights into hippocampal organization, as in this study, can elucidate mechanisms underlying motivational behaviors, spatial navigation, and memory formation.

Learning-related changes in cellular activity within mouse dentate gyrus during trace eyeblink conditioning.

Because the dentate gyrus serves as the first site for information processing in the hippocampal trisynaptic circuit, it an important structure for the formation of associative memories. Previous findings in rabbit had recorded populations of cells within dentate gyrus that may bridge the temporal gap between stimuli to support memory formation during trace eyeblink conditioning, an associative learning task. However, this previous work was unable to identify the types of cells demonstrating this type of activity. To explore these changes further, we did in vivo single-neuron recording in conjunction with physiological determination of cell types to investigate the functional role of granule cells, mossy cells, and interneurons in dentate gyrus during learning. Tetrode recordings were performed in young-adult mice during training on trace eyeblink conditioning, a hippocampal-dependent temporal associative memory task. Conditioned mice were able to successfully learn the task, with male mice learning at a faster rate than female mice. In the conditioned group, granule cells tended to show an increase in firing rate during conditioned stimulus presentation while mossy cells showed a decrease in firing rate during the trace interval and the unconditioned stimulus. Interestingly, populations of interneurons demonstrated learning-related increases and decreases in activity that began at onset of the conditioned stimulus and persisted through the trace interval. The current study also found a significant increase in theta power during stimuli presentation in conditioned animals, and this change in theta decreased over time. Ultimately, these data suggest unique involvement of granule cells, mossy cells, and interneurons in dentate gyrus in the formation of a trace associative memory. This work expands our knowledge of dentate gyrus function, helping to discern how aging and disease might disrupt this process.

Sex-Dependent Effects of Chronic Microdrive Implantation on Acquisition of Trace Eyeblink Conditioning.

Neuroscience techniques, including in vivo recording, have allowed for a great expansion in knowledge; however, this technology may also affect the very phenomena researchers set out to investigate. Including both female and male mice in our associative learning experiments shed light on sex differences on the impact of chronic implantation of tetrodes on learning. While previous research showed intact female mice acquired trace eyeblink conditioning faster than male and ovariectomized females, implantation of chronic microdrive arrays showed sexually dimorphic effects on learning. Microdrive implanted male mice acquired the associative learning paradigm faster than both intact and ovariectomized females. These effects were not due to the weight of the drive alone, as there were no significant sex-differences in learning of animals that received "dummy drive" implants without tetrodes lowered into the brain. Tandem mass tag mass spectrometry and western blot analysis suggest that significant alterations in the MAPK pathway, acute inflammation, and brain derived neurotrophic factor may underlie these observed sex- and surgery-dependent effects on learning.

Intrinsic Excitability Increase in Cerebellar Purkinje Cells after Delay Eye-Blink Conditioning in Mice.

Cerebellar-based learning is thought to rely on synaptic plasticity, particularly at synaptic inputs to Purkinje cells. Recently, however, other complementary mechanisms have been identified. Intrinsic plasticity is one such mechanism, and depends in part on the downregulation of calcium-dependent SK-type K+ channels, which contribute to a medium-slow afterhyperpolarization (AHP) after spike bursts, regulating membrane excitability. In the hippocampus, intrinsic plasticity plays a role in trace eye-blink conditioning; however, corresponding excitability changes in the cerebellum in associative learning, such as in trace or delay eye-blink conditioning, are less well studied. Whole-cell patch-clamp recordings were obtained from Purkinje cells in cerebellar slices prepared from male mice ∼48 h after they learned a delay eye-blink conditioning task. Over a period of repeated training sessions, mice received either paired trials of a tone coterminating with a periorbital shock (conditioning) or trials in which these stimuli were randomly presented in an unpaired manner (pseudoconditioning). Purkinje cells from conditioned mice show a significantly reduced AHP after trains of parallel fiber stimuli and after climbing fiber evoked complex spikes. The number of spikelets in the complex spike waveform is increased after conditioning. Moreover, we find that SK-dependent intrinsic plasticity is occluded in conditioned, but not pseudoconditioned mice. These findings show that excitability is enhanced in Purkinje cells after delay eye-blink conditioning, and point toward a downregulation of SK channels as a potential underlying mechanism. The observation that this learning effect lasts at least up to 2 d after training shows that intrinsic plasticity regulates excitability in the long term.SIGNIFICANCE STATEMENT Plasticity of membrane excitability ("intrinsic plasticity") has been observed in invertebrate and vertebrate neurons, coinduced with synaptic plasticity or in isolation. Although the cellular phenomenon per se is well established, it remains unclear what role intrinsic plasticity plays in learning and if it even persists long enough to serve functions in engram physiology beyond aiding synaptic plasticity. Here, we demonstrate that cerebellar Purkinje cells upregulate excitability in delay eye-blink conditioning, a form of motor learning. This plasticity is observed 48 h after training and alters synaptically evoked spike firing and integrative properties of these neurons. These findings show that intrinsic plasticity enhances the spike firing output of Purkinje cells and persists over the course of days.

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.

Differential responsivity of neurons in perirhinal cortex, lateral entorhinal cortex, and dentate gyrus during time-bridging learning.

Many studies have focused on the function of hippocampal region CA1 as a critical site for associative memory, but much less is known about changes in the afferents to CA1. Here we report the activity of multiple single neurons from perirhinal and entorhinal cortex and from dentate gyrus during trace eyeblink conditioning as well as consolidated recall, and in pseudo-conditioned control rabbits. We also report an analysis of theta activity filtered from the local field potential (LFP). Our results show early associative changes in single-neuron firing rate as well as theta oscillations in lateral entorhinal cortex (EC) and dentate gyrus (DG), and increases in the number of responsive neurons in perirhinal cortex. In both EC and DG, a subset of neurons from conditioned animals exhibited an elevated baseline firing rate and large responses to the conditioned stimulus and trace period. A similar population of cells has been seen in DG and in medial, but not lateral, EC during spatial tasks, suggesting that lateral EC contains cells responsive to a temporal associative task. In contrast to recent studies in our laboratory that found significant CA1 contributions to long-term memory, the activity profiles of neurons within EC and DG were similar for conditioned and pseudoconditioned rabbits during post-consolidation sessions. Collectively these results demonstrate that individual subregions of medial temporal lobe differentially support new and remotely acquired memories. Neuron firing profiles were similar on training trials when conditioned responses were and were not exhibited, demonstrating that these temporal lobe regions represent the CS-US association and do not control the behavioral response. The analysis of theta activity revealed that theta power was modulated by the conditioning stimuli in both the conditioned and pseudoconditioned groups and that although both groups exhibited a resetting of phase to the corneal airpuff, only the conditioned group exhibited a resetting of phase to the whisker conditioned stimulus.

Store depletion-induced h-channel plasticity rescues a channelopathy linked to Alzheimer's disease.

Voltage-gated ion channels are critical for neuronal integration. Some of these channels, however, are misregulated in several neurological disorders, causing both gain- and loss-of-function channelopathies in neurons. Using several transgenic mouse models of Alzheimer's disease (AD), we find that sub-threshold voltage signals strongly influenced by hyperpolarization-activated, cyclic nucleotide-gated (HCN) channels progressively deteriorate over chronological aging in hippocampal CA1 pyramidal neurons. The degraded signaling via HCN channels in the transgenic mice is accompanied by an age-related global loss of their non-uniform dendritic expression. Both the aberrant signaling via HCN channels and their mislocalization could be restored using a variety of pharmacological agents that target the endoplasmic reticulum (ER). Our rescue of the HCN channelopathy helps provide molecular details into the favorable outcomes of ER-targeting drugs on the pathogenesis and synaptic/cognitive deficits in AD mouse models, and implies that they might have beneficial effects on neurological disorders linked to HCN channelopathies.

Dendritic spinopathy in transgenic mice expressing ALS/dementia-linked mutant UBQLN2.

Mutations in the gene encoding ubiquilin2 (UBQLN2) cause amyotrophic lateral sclerosis (ALS), frontotemporal type of dementia, or both. However, the molecular mechanisms are unknown. Here, we show that ALS/dementia-linked UBQLN2(P497H) transgenic mice develop neuronal pathology with ubiquilin2/ubiquitin/p62-positive inclusions in the brain, especially in the hippocampus, recapitulating several key pathological features of dementia observed in human patients with UBQLN2 mutations. A major feature of the ubiquilin2-related pathology in these mice, and reminiscent of human disease, is a dendritic spinopathy with protein aggregation in the dendritic spines and an associated decrease in dendritic spine density and synaptic dysfunction. Finally, we show that the protein inclusions in the dendritic spines are composed of several components of the proteasome machinery, including Ub(G76V)-GFP, a representative ubiquitinated protein substrate that is accumulated in the transgenic mice. Our data, therefore, directly link impaired protein degradation to inclusion formation that is associated with synaptic dysfunction and cognitive deficits. These data imply a convergent molecular pathway involving synaptic protein recycling that may also be involved in other neurodegenerative disorders, with implications for development of widely applicable rational therapeutics.

Pretrial functional connectivity differentiates behavioral outcomes during trace eyeblink conditioning in the rabbit.

Fluctuations in neural activity can produce states that facilitate and accelerate task-related performance. Acquisition of trace eyeblink conditioning (tEBC) in the rabbit is enhanced when trials are contingent on optimal pretrial activity in the hippocampus. Other regions which are essential for whisker-signaled tEBC, such as the cerebellar interpositus nucleus (IPN), somatosensory and prelimbic cortices, may also show optimal connectivity prior to successful performance. Functional magnetic resonance imaging (fMRI) was acquired in nine rabbits during tEBC on the first and tenth days of initial training and once again after a 30-d, training-free hiatus. Data acquired during the intertrial interval was parsed depending on whether or not a conditioned response (CR) occurred on the upcoming trial and seed-based functional connectivity was calculated among the IPN, hippocampus, somatosensory, and prelimbic cortices. Functional connectivity between the left somatosensory cortex and right IPN, regions critical for establishing and producing CRs evoked by right vibrissae vibration and right corneal airpuff, was significantly negative prior to successful, CR trials as compared with unsuccessful, non-CR trials. Differences were not observed for any of the other possible combinations of connectivity. Our results demonstrate that specific pretrial functional connectivity exists within the rabbit brain and differentiates between upcoming behavioral response outcomes. Online analysis of network fluctuations has the potential to be used as the basis for therapeutic interventions to facilitate learning and memory.

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.

Activity-induced manganese-dependent MRI (AIM-MRI) and functional MRI in awake rabbits during somatosensory stimulation.

Activity-induced manganese-dependent MRI (AIM-MRI) is a powerful tool to track system-wide neural activity using high resolution, quantitative T1-weighted MRI in animal models and has significant advantages for investigating neural activity over other modalities including BOLD fMRI. With AIM-MRI, Mn(2+) ions enter neurons via voltage-gated calcium channels preferentially active during the time of experimental exposure. A broad range of AIM-MRI studies using different species studying different phenomena have been performed, but few of these studies provide a systematic evaluation of the factors influencing the detection of Mn(2+) such as dosage and the temporal characteristics of Mn(2+) uptake. We identified an optimal dose of Mn(2+) (25 mg/kg, s.c.) in order to characterize the time-course of Mn(2+) accumulation in active neural regions in the rabbit. T1-weighted MRI and functional MRI were collected 0-3, 6-9, and 24-27 h post-Mn(2+) injection while the vibrissae on the right side were vibrated. Significant BOLD activation in the left somatosensory (SS) cortex and left ventral posteromedial (VPM) thalamic nucleus was detected during whisker vibration. T1-weighted signal intensities were extracted from these regions, their corresponding contralateral regions and the visual cortex (to serve as controls). A significant elevation in T1-weighted signal intensity in the left SS cortex (relative to right) was evident 6-9 and 24-27 h post-Mn(2+) injection while the left VPM thalamus showed a significant enhancement (relative to the right) only during the 24-27 h session. Visual cortex showed no hemispheric difference at any timepoint. Our results suggest that studies employing AIM-MRI would benefit by conducting experimental manipulations 6-24 h after subcutaneous MnCl2 injections to optimize the concentration of contrast agent in the regions active during the exposure.

Intrinsic connectivity of neural networks in the awake rabbit.

The way in which the brain is functionally connected into different networks has emerged as an important research topic in order to understand normal neural processing and signaling. Since some experimental manipulations are difficult or unethical to perform in humans, animal models are better suited to investigate this topic. Rabbits are a species that can undergo MRI scanning in an awake and conscious state with minimal preparation and habituation. In this study, we characterized the intrinsic functional networks of the resting New Zealand White rabbit brain using BOLD fMRI data. Group independent component analysis revealed seven networks similar to those previously found in humans, non-human primates and/or rodents including the hippocampus, default mode, cerebellum, thalamus, and visual, somatosensory, and parietal cortices. For the first time, the intrinsic functional networks of the resting rabbit brain have been elucidated demonstrating the rabbit's applicability as a translational animal model. Without the confounding effects of anesthetics or sedatives, future experiments may employ rabbits to understand changes in neural connectivity and brain functioning as a result of experimental manipulation (e.g., temporary or permanent network disruption, learning-related changes, and drug administration).

Functional reorganization of a prefrontal cortical network mediating consolidation of trace eyeblink conditioning.

The medial prefrontal cortex (mPFC) has been studied for its role in various cognitive functions, but the roles of its subregions remain unclear. We performed tetrode recordings simultaneously from prelimbic (PL) and rostral (rACC) and caudal (cACC) anterior cingulate subregions of the rabbit mPFC to understand their interactions during learning and tests of remote memory retention for whisker-signaled trace eyeblink conditioning. cACC neurons exhibited an innate response to the conditioning stimulus (CS) that rapidly decreased across sessions, suggesting an attentional role for facilitating CS-US associations. rACC neurons from conditioned rabbits exhibited robust responses to the CS that decreased within each session, possibly evaluating its emotional salience. PL neurons exhibited robust persistent activity during the trace interval during tests of remote memory retention, suggesting its involvement in retrieval and execution of a consolidated response. Mechanistically, conditioning was associated with a greater percentage of persistently responsive neurons than neurons from pseudoconditioned control rabbits, and responses differed significantly between trials with and without conditioned responses. Collectively, these responses reflect a functional reorganization of neural activity within the prefrontal network from an attentional mode to one that orchestrates the retrieval and execution of the learned response.

Robust hippocampal responsivity during retrieval of consolidated associative memory.

A contentious point in memory research is whether or not the hippocampus plays a time-limited role in the consolidation of declarative memories. A widely held view is that declarative memories are initially encoded in the hippocampus, then transferred to the neocortex for long-term storage. Alternate views argue instead that the hippocampus continues to play a role in remote memory recall. These competing theories are largely based on human amnesic and animal lesion/inactivation studies. However, in vivo electrophysiological evidence supporting these views is scarce. Given that other studies examining the role of the hippocampus in remote memory retrieval using lesion and imaging techniques in human and animal models have provided mixed results, it would be particularly useful to gain insight at the in vivo electrophysiological level. Here we report hippocampal single-neuron and theta activity recorded longitudinally during acquisition and remote retrieval of trace eyeblink conditioning. Results from conditioned rabbits were compared to those obtained from yoked pseudo-conditioned control rabbits. Results reveal continued learning-specific hippocampal activity one month after initial acquisition of the task. Our findings yield insight into the normal physiological responses of the hippocampus during memory processes and provide compelling in vivo electrophysiological evidence that the hippocampus is involved in both acquisition and retrieval of consolidated memories.

Caudate nucleus in retrieval of trace eyeblink conditioning after consolidation.

Trace eyeblink conditioning (EBC) is an associative learning task in which a stimulus-free trace period separates the presentation of a behaviorally neutral conditioned stimulus (CS; whisker stimulation) from a behaviorally salient unconditioned stimulus (US; air puff to the eye). Repeated pairings of the CS with the US results in the emergence of the conditioned response (CR; eyeblink after CS presentation and before US presentation). The goal of these experiments was to determine whether the caudate nucleus (CN) plays a role in retrieval of previously acquired trace EBC after memory consolidation. Lesions of the CN were made 1 month after initial trace EBC. CN-lesioned rabbits performed fewer adaptive CRs and more short-latency non-adaptive responses than sham-lesioned controls. They were not able to improve their CR performance after consolidation as were controls. Single-unit recordings taken from separate cohorts of rabbits demonstrated that neurons in the CN were still responsive to the CS and US 1 month after initial trace EBC, particularly in the medial and ventral CN on trials when a CR occurred. The proportion of rate-increasing neurons was higher in trace-conditioned than in pseudo-conditioned rabbits. Neurons in regions destroyed in the behavioral experiment demonstrated prolonged firing during the trace period, which might underlie the results from the behavioral experiment. These data demonstrate that the CN plays an important role in retrieval of a previously learned associative task after memory consolidation has occurred.

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.

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.