Transient Seizure Clusters and Epileptiform Activity Following Widespread Bilateral Hippocampal Interneuron Ablation.

Interneuron loss is a prominent feature of temporal lobe epilepsy in both animals and humans and is hypothesized to be critical for epileptogenesis. As loss occurs concurrently with numerous other potentially proepileptogenic changes, however, the impact of interneuron loss in isolation remains unclear. For the present study, we developed an intersectional genetic approach to induce bilateral diphtheria toxin-mediated deletion of Vgat-expressing interneurons from dorsal and ventral hippocampus. In a separate group of mice, the same population was targeted for transient neuronal silencing with DREADDs. Interneuron ablation produced dramatic seizure clusters and persistent epileptiform activity. Surprisingly, after 1 week seizure activity declined precipitously and persistent epileptiform activity disappeared. Occasional seizures (≈1/day) persisted to the end of the experiment at 4 weeks. In contrast to the dramatic impact of interneuron ablation, transient silencing produced large numbers of interictal spikes, a significant but modest increase in seizure occurrence and changes in EEG frequency band power. Taken together, findings suggest that the hippocampus regains relative homeostasis-with occasional breakthrough seizures-in the face of an extensive and abrupt loss of interneurons.

Neurovascular Development in Pten and Tsc2 Mouse Mutants.

Hyperactivation of the mechanistic target of rapamycin (mTOR) signaling pathway is linked to more than a dozen neurologic diseases, causing a range of pathologies, including excess neuronal growth, disrupted neuronal migration, cortical dysplasia, epilepsy and autism. The mTOR pathway also regulates angiogenesis. For the present study, therefore, we queried whether loss of Pten or Tsc2, both mTOR negative regulators, alters brain vasculature in three mouse models: one with Pten loss restricted to hippocampal dentate granule cells [DGC-Pten knock-outs (KOs)], a second with widespread Pten loss from excitatory forebrain neurons (FB-Pten KOs) and a third with focal loss of Tsc2 from cortical excitatory neurons (f-Tsc2 KOs). Total hippocampal vessel length and volume per dentate gyrus were dramatically increased in DGC-Pten knock-outs. DGC-Pten knock-outs had larger dentate gyri overall, however, and when normalized to these larger structures, vessel density was preserved. In addition, tests of blood-brain barrier integrity did not reveal increased permeability. FB-Pten KOs recapitulated the findings in the more restricted DGC-Pten KOs, with increased vessel area, but preserved vessel density. FB-Pten KOs did, however, exhibit elevated levels of the angiogenic factor VegfA. In contrast to findings with Pten, focal loss of Tsc2 from cortical excitatory neurons produced a localized increase in vessel density. Together, these studies demonstrate that hypervascularization is not a consistent feature of mTOR hyperactivation models and suggest that loss of different mTOR pathway regulatory genes exert distinct effects on angiogenesis.

Hippocampal glucocorticoid receptors modulate status epilepticus severity.

Status epilepticus (SE) is a life-threatening medical emergency with significant morbidity and mortality. SE is associated with a robust and sustained increase in serum glucocorticoids, reaching concentrations sufficient to activate the dense population of glucocorticoid receptors (GRs) expressed among hippocampal excitatory neurons. Glucocorticoid exposure can increase hippocampal neuron excitability; however, whether activation of hippocampal GRs during SE exacerbates seizure severity remains unknown. To test this, a viral strategy was used to delete GRs from a subset of hippocampal excitatory neurons in adult male and female mice, producing hippocampal GR knockdown mice. Two weeks after GR knockdown, mice were challenged with the convulsant drug pilocarpine to induce SE. GR knockdown had opposing effects on early vs late seizure behaviors, with sex influencing responses. For both male and female mice, the onset of mild behavioral seizures was accelerated by GR knockdown. In contrast, GR knockdown delayed the onset of more severe convulsive seizures and death in male mice. Concordantly, GR knockdown also blunted the SE-induced rise in serum corticosterone in male mice. GR knockdown did not alter survival times or serum corticosterone in females. To assess whether loss of GR affected susceptibility to SE-induced cell death, within-animal analyses were conducted comparing local GR knockdown rates to local cell loss. GR knockdown did not affect the degree of localized neuronal loss, suggesting cell-intrinsic GR signaling neither protects nor sensitizes neurons to acute SE-induced death. Overall, the findings reveal that hippocampal GRs exert an anti-convulsant role in both males and females in the early stages of SE, followed by a switch to a pro-convulsive role for males only. Findings reveal an unexpected complexity in the interaction between hippocampal GR activation and the progression of SE.

Spatial and Temporal Comparisons of Calcium Channel and Intrinsic Signal Imaging During in Vivo Cortical Spreading Depolarizations in Healthy and Hypoxic Brains.

BACKGROUND: Spreading depolarizations (SDs) can be viewed at a cellular level using calcium imaging (CI), but this approach is limited to laboratory applications and animal experiments. Optical intrinsic signal imaging (OISI), on the other hand, is amenable to clinical use and allows viewing of large cortical areas without contrast agents. A better understanding of the behavior of OISI-observed SDs under different brain conditions is needed. METHODS: We performed simultaneous calcium and OISI of SDs in GCaMP6f mice. SDs propagate through the cortex as a pathological wave and trigger a neurovascular response that can be imaged with both techniques. We imaged both mechanically stimulated SDs (sSDs) in healthy brains and terminal SDs (tSDs) induced by system hypoxia and cardiopulmonary failure. RESULTS: We observed a lag in the detection of SDs in the OISI channels compared with CI. sSDs had a faster velocity than tSDs, and tSDs had a greater initial velocity for the first 400 µm when observed with CI compared with OISI. However, both imaging methods revealed similar characteristics, including a decrease in the sSD (but not tSD) velocities as the wave moved away from the site of initial detection. CI and OISI also showed similar spatial propagation of the SD throughout the image field. Importantly, only OISI allowed regional ischemia to be detected before tSDs occurred. CONCLUSIONS: Altogether, data indicate that monitoring either neural activity or intrinsic signals with high-resolution optical imaging can be useful to assess SDs, but OISI may be a clinically applicable way to predict, and therefore possibly mitigate, hypoxic-ischemic tSDs.

mTOR-driven neural circuit changes initiate an epileptogenic cascade.

Mutations in genes regulating mTOR pathway signaling are now recognized as a significant cause of epilepsy. Interestingly, these mTORopathies are often caused by somatic mutations, affecting variable numbers of neurons. To better understand how this variability affects disease phenotype, we developed a mouse model in which the mTOR pathway inhibitor Pten can be deleted from 0 to 40 % of hippocampal granule cells. In vivo, low numbers of knockout cells caused focal seizures, while higher numbers led to generalized seizures. Generalized seizures coincided with the loss of local circuit interneurons. In hippocampal slices, low knockout cell loads produced abrupt reductions in population spike threshold, while spontaneous excitatory postsynaptic currents and circuit level recurrent activity increased gradually with rising knockout cell load. Findings demonstrate that knockout cells load is a critical variable regulating disease phenotype, progressing from subclinical circuit abnormalities to electrobehavioral seizures with secondary involvement of downstream neuronal populations.

Impact of mTOR hyperactive neurons on the morphology and physiology of adjacent neurons: Do PTEN KO cells make bad neighbors?

Hyperactivation of the mechanistic target of rapamycin (mTOR) pathway is associated with epilepsy, autism and brain growth abnormalities in humans. mTOR hyperactivation often results from developmental somatic mutations, producing genetic lesions and associated dysfunction in relatively restricted populations of neurons. Disrupted brain regions, such as those observed in focal cortical dysplasia, can contain a mix of normal and mutant cells. Mutant cells exhibit robust anatomical and physiological changes. Less clear, however, is whether adjacent, initially normal cells are affected by the presence of abnormal cells. To explore this question, we used a conditional, inducible mouse model approach to delete the mTOR negative regulator phosphatase and tensin homolog (PTEN) from <1% to >30% of hippocampal dentate granule cells. We then examined the morphology of PTEN-expressing granule cells located in the same dentate gyri as the knockout (KO) cells. Despite the development of spontaneous seizures in higher KO animals, and disease worsening with increasing age, the morphology and physiology of PTEN-expressing cells was only modestly affected. PTEN-expressing cells had smaller somas than cells from control animals, but other parameters were largely unchanged. These findings contrast with the behavior of PTEN KO cells, which show increasing dendritic extent with greater KO cell load. Together, the findings indicate that genetically normal neurons can exhibit relatively stable morphology and intrinsic physiology in the presence of nearby pathological neurons and systemic disease.

Self-reinforcing effects of mTOR hyperactive neurons on dendritic growth.

Loss of the mTOR pathway negative regulator PTEN from hippocampal dentate granule cells leads to neuronal hypertrophy, increased dendritic branching and aberrant basal dendrite formation in animal models. Similar changes are evident in humans with mTOR pathway mutations. These genetic conditions are associated with autism, cognitive dysfunction and epilepsy. Interestingly, humans with mTOR pathway mutations often present with mosaic disruptions of gene function, producing lesions that range from focal cortical dysplasia to hemimegalanecephaly. Whether mTOR-mediated neuronal dysmorphogenesis is impacted by the number of affected cells, however, is not known. mTOR mutations can produce secondary comorbidities, including brain hypertrophy and seizures, which could exacerbate dysmorphogenesis among mutant cells. To determine whether the percentage or "load" of PTEN knockout granule cells impacts the morphological development of these same cells, we generated two groups of PTEN knockout mice. In the first, PTEN deletion rates were held constant, at about 5%, and knockout cell growth over time was assessed. Knockout cells exhibited significant dendritic growth between 7 and 18 weeks, demonstrating that aberrant dendritic growth continues even after the cells reach maturity. In the second group of mice, PTEN was deleted from 2 to 37% of granule cells to determine whether deletion rate was a factor in driving this continued growth. Multivariate analysis revealed that both age and knockout cell load contributed to knockout cell dendritic growth. Although the mechanism remains to be determined, these findings demonstrate that large numbers of mutant neurons can produce self-reinforcing effects on their own growth.

PTEN deletion increases hippocampal granule cell excitability in male and female mice.

Deletion of the mTOR pathway inhibitor PTEN from postnatally-generated hippocampal dentate granule cells causes epilepsy. Here, we conducted field potential, whole cell recording and single cell morphology studies to begin to elucidate the mechanisms by which granule cell-specific PTEN-loss produces disease. Cells from both male and female mice were recorded to identify sex-specific effects. PTEN knockout granule cells showed altered intrinsic excitability, evident as a tendency to fire in bursts. PTEN knockout granule cells also exhibited increased frequency of spontaneous excitatory synaptic currents (sEPSCs) and decreased frequency of inhibitory currents (sIPSCs), further indicative of a shift towards hyperexcitability. Morphological studies of PTEN knockout granule cells revealed larger dendritic trees, more dendritic branches and an impairment of dendrite self-avoidance. Finally, cells from both female control and female knockout mice received more sEPSCs and more sIPSCs than corresponding male cells. Despite the difference, the net effect produced statistically equivalent EPSC/IPSC ratios. Consistent with this latter observation, extracellularly evoked responses in hippocampal slices were similar between male and female knockouts. Both groups of knockouts were abnormal relative to controls. Together, these studies reveal a host of physiological and morphological changes among PTEN knockout cells likely to underlie epileptogenic activity. SIGNIFICANCE STATEMENT: Hyperactivation of the mTOR pathway is associated with numerous neurological diseases, including autism and epilepsy. Here, we demonstrate that deletion of the mTOR negative regulator, PTEN, from a subset of hippocampal dentate granule impairs dendritic patterning, increases excitatory input and decreases inhibitory input. We further demonstrate that while granule cells from female mice receive more excitatory and inhibitory input than males, PTEN deletion produces mostly similar changes in both sexes. Together, these studies provide new insights into how the relatively small number (≈200,000) of PTEN knockout granule cells instigates the development of the profound epilepsy syndrome evident in both male and female animals in this model.

Disrupted hippocampal network physiology following PTEN deletion from newborn dentate granule cells.

Abnormal hippocampal granule cells are present in patients with temporal lobe epilepsy, and are a prominent feature of most animal models of the disease. These abnormal cells are hypothesized to contribute to epileptogenesis. Isolating the specific effects of abnormal granule cells on hippocampal physiology, however, has been difficult in traditional temporal lobe epilepsy models. While epilepsy induction in these models consistently produces abnormal granule cells, the causative insults also induce widespread cell death among hippocampal, cortical and subcortical structures. Recently, we demonstrated that introducing morphologically abnormal granule cells into an otherwise normal mouse brain - by selectively deleting the mTOR pathway inhibitor PTEN from postnatally-generated granule cells - produced hippocampal and cortical seizures. Here, we conducted acute slice field potential recordings to assess the impact of these cells on hippocampal function. PTEN deletion from a subset of granule cells reproduced aberrant responses present in traditional epilepsy models, including enhanced excitatory post-synaptic potentials (fEPSPs) and multiple, rather than single, population spikes in response to perforant path stimulation. These findings provide new evidence that abnormal granule cells initiate a process of epileptogenesis - in the absence of widespread cell death - which culminates in an abnormal dentate network similar to other models of temporal lobe epilepsy. Findings are consistent with the hypothesis that accumulation of abnormal granule cells is a common mechanism of temporal lobe epileptogenesis.

Impact of rapamycin on status epilepticus induced hippocampal pathology and weight gain.

Growing evidence implicates the dentate gyrus in temporal lobe epilepsy (TLE). Dentate granule cells limit the amount of excitatory signaling through the hippocampus and exhibit striking neuroplastic changes that may impair this function during epileptogenesis. Furthermore, aberrant integration of newly-generated granule cells underlies the majority of dentate restructuring. Recently, attention has focused on the mammalian target of rapamycin (mTOR) signaling pathway as a potential mediator of epileptogenic change. Systemic administration of the mTOR inhibitor rapamycin has promising therapeutic potential, as it has been shown to reduce seizure frequency and seizure severity in rodent models. Here, we tested whether mTOR signaling facilitates abnormal development of granule cells during epileptogenesis. We also examined dentate inflammation and mossy cell death in the dentate hilus. To determine if mTOR activation is necessary for abnormal granule cell development, transgenic mice that harbored fluorescently-labeled adult-born granule cells were treated with rapamycin following pilocarpine-induced status epilepticus. Systemic rapamycin effectively blocked phosphorylation of S6 protein (a readout of mTOR activity) and reduced granule cell mossy fiber axon sprouting. However, the accumulation of ectopic granule cells and granule cells with aberrant basal dendrites was not significantly reduced. Mossy cell death and reactive astrocytosis were also unaffected. These data suggest that anti-epileptogenic effects of mTOR inhibition may be mediated by mechanisms other than inhibition of these common dentate pathologies. Consistent with this conclusion, rapamycin prevented pathological weight gain in epileptic mice, suggesting that rapamycin might act on central circuits or even peripheral tissues controlling weight gain in epilepsy.

PTEN deletion from adult-generated dentate granule cells disrupts granule cell mossy fiber axon structure.

Dysregulation of the mTOR-signaling pathway is implicated in the development of temporal lobe epilepsy. In mice, deletion of PTEN from hippocampal dentate granule cells leads to mTOR hyperactivation and promotes the rapid onset of spontaneous seizures. The mechanism by which these abnormal cells initiate epileptogenesis, however, is unclear. PTEN-knockout granule cells develop abnormally, exhibiting morphological features indicative of increased excitatory input. If these cells are directly responsible for seizure genesis, it follows that they should also possess increased output. To test this prediction, dentate granule cell axon morphology was quantified in control and PTEN-knockout mice. Unexpectedly, PTEN deletion increased giant mossy fiber bouton spacing along the axon length, suggesting reduced innervation of CA3. Increased width of the mossy fiber axon pathway in stratum lucidum, however, which likely reflects an unusual increase in mossy fiber axon collateralization in this region, offsets the reduction in boutons per axon length. These morphological changes predict a net increase in granule cell innervation of CA3. Increased diameter of axons from PTEN-knockout cells would further enhance granule cell communication with CA3. Altogether, these findings suggest that amplified information flow through the hippocampal circuit contributes to seizure occurrence in the PTEN-knockout mouse model of temporal lobe epilepsy.

Clonal Analysis of Newborn Hippocampal Dentate Granule Cell Proliferation and Development in Temporal Lobe Epilepsy.

Hippocampal dentate granule cells are among the few neuronal cell types generated throughout adult life in mammals. In the normal brain, new granule cells are generated from progenitors in the subgranular zone and integrate in a typical fashion. During the development of epilepsy, granule cell integration is profoundly altered. The new cells migrate to ectopic locations and develop misoriented "basal" dendrites. Although it has been established that these abnormal cells are newly generated, it is not known whether they arise ubiquitously throughout the progenitor cell pool or are derived from a smaller number of "bad actor" progenitors. To explore this question, we conducted a clonal analysis study in mice expressing the Brainbow fluorescent protein reporter construct in dentate granule cell progenitors. Mice were examined 2 months after pilocarpine-induced status epilepticus, a treatment that leads to the development of epilepsy. Brain sections were rendered translucent so that entire hippocampi could be reconstructed and all fluorescently labeled cells identified. Our findings reveal that a small number of progenitors produce the majority of ectopic cells following status epilepticus, indicating that either the affected progenitors or their local microenvironments have become pathological. By contrast, granule cells with "basal" dendrites were equally distributed among clonal groups. This indicates that these progenitors can produce normal cells and suggests that global factors sporadically disrupt the dendritic development of some new cells. Together, these findings strongly predict that distinct mechanisms regulate different aspects of granule cell pathology in epilepsy.

Mechanisms regulating neuronal excitability and seizure development following mTOR pathway hyperactivation.

The phosphatidylinositol-3-kinase/phosphatase and tensin homolog (PTEN)-mammalian target of rapamycin (mTOR) pathway regulates a variety of neuronal functions, including cell proliferation, survival, growth, and plasticity. Dysregulation of the pathway is implicated in the development of both genetic and acquired epilepsies. Indeed, several causal mutations have been identified in patients with epilepsy, the most prominent of these being mutations in PTEN and tuberous sclerosis complexes 1 and 2 (TSC1, TSC2). These genes act as negative regulators of mTOR signaling, and mutations lead to hyperactivation of the pathway. Animal models deleting PTEN, TSC1, and TSC2 consistently produce epilepsy phenotypes, demonstrating that increased mTOR signaling can provoke neuronal hyperexcitability. Given the broad range of changes induced by altered mTOR signaling, however, the mechanisms underlying seizure development in these animals remain uncertain. In transgenic mice, cell populations with hyperactive mTOR have many structural abnormalities that support recurrent circuit formation, including somatic and dendritic hypertrophy, aberrant basal dendrites, and enlargement of axon tracts. At the functional level, mTOR hyperactivation is commonly, but not always, associated with enhanced synaptic transmission and plasticity. Moreover, these populations of abnormal neurons can affect the larger network, inducing secondary changes that may explain paradoxical findings reported between cell and network functioning in different models or at different developmental time points. Here, we review the animal literature examining the link between mTOR hyperactivation and epileptogenesis, emphasizing the impact of enhanced mTOR signaling on neuronal form and function.

Age-related changes in rostral basal forebrain cholinergic and GABAergic projection neurons: relationship with spatial impairment.

Both cholinergic and GABAergic projections from the rostral basal forebrain contribute to hippocampal function and mnemonic abilities. While dysfunction of cholinergic neurons has been heavily implicated in age-related memory decline, significantly less is known regarding how age-related changes in codistributed GABAergic projection neurons contribute to a decline in hippocampal-dependent spatial learning. In the current study, confocal stereology was used to quantify cholinergic (choline acetyltransferase [ChAT] immunopositive) neurons, GABAergic projection (glutamic decarboxylase 67 [GAD67] immunopositive) neurons, and total (neuronal nuclei [NeuN] immunopositive) neurons in the rostral basal forebrain of young and aged rats that were first characterized on a spatial learning task. ChAT immunopositive neurons were significantly but modestly reduced in aged rats. Although ChAT immunopositive neuron number was strongly correlated with spatial learning abilities among young rats, the reduction of ChAT immunopositive neurons was not associated with impaired spatial learning in aged rats. In contrast, the number of GAD67 immunopositive neurons was robustly and selectively elevated in aged rats that exhibited impaired spatial learning. Interestingly, the total number of rostral basal forebrain neurons was comparable in young and aged rats, regardless of their cognitive status. These data demonstrate differential effects of age on phenotypically distinct rostral basal forebrain projection neurons, and implicate dysregulated cholinergic and GABAergic septohippocampal circuitry in age-related mnemonic decline.

Excessive activation of mTOR in postnatally generated granule cells is sufficient to cause epilepsy.

The dentate gyrus is hypothesized to function as a "gate," limiting the flow of excitation through the hippocampus. During epileptogenesis, adult-generated granule cells (DGCs) form aberrant neuronal connections with neighboring DGCs, disrupting the dentate gate. Hyperactivation of the mTOR signaling pathway is implicated in driving this aberrant circuit formation. While the presence of abnormal DGCs in epilepsy has been known for decades, direct evidence linking abnormal DGCs to seizures has been lacking. Here, we isolate the effects of abnormal DGCs using a transgenic mouse model to selectively delete PTEN from postnatally generated DGCs. PTEN deletion led to hyperactivation of the mTOR pathway, producing abnormal DGCs morphologically similar to those in epilepsy. Strikingly, animals in which PTEN was deleted from ≥ 9% of the DGC population developed spontaneous seizures in about 4 weeks, confirming that abnormal DGCs, which are present in both animals and humans with epilepsy, are capable of causing the disease.

GABA(B) receptor GTP-binding is decreased in the prefrontal cortex but not the hippocampus of aged rats.

Gamma aminobutyric acid (GABA)(B) receptors (GABA(B)Rs) have been linked to a wide range of physiological and cognitive processes and are of interest for treating a number of neurodegenerative and psychiatric disorders. As many of these diseases are associated with advanced age, it is important to understand how the normal aging process impacts GABA(B)R expression and signaling. Thus, we investigated GABA(B)R expression and function in the prefrontal cortex (PFC) and hippocampus of young and aged rats characterized in a spatial learning task. Baclofen-stimulated GTP-binding and GABA(B)R1 and GABA(B)R2 proteins were reduced in the prefrontal cortex of aged rats but these reductions were not associated with spatial learning abilities. In contrast, hippocampal GTP-binding was comparable between young and aged rats but reduced hippocampal GABA(B)R1 expression was observed in aged rats with spatial learning impairment. These data demonstrate marked regional differences in GABA(B)R complexes in the adult and aged brain and could have implications for both understanding the role of GABAergic processes in normal brain function and the development of putative interventions that target this system.

Dopaminergic modulation of risky decision-making.

Many psychiatric disorders are characterized by abnormal risky decision-making and dysregulated dopamine receptor expression. The current study was designed to determine how different dopamine receptor subtypes modulate risk-taking in young adult rats, using a "Risky Decision-making Task" that involves choices between small "safe" rewards and large "risky" rewards accompanied by adverse consequences. Rats showed considerable, stable individual differences in risk preference in the task, which were not related to multiple measures of reward motivation, anxiety, or pain sensitivity. Systemic activation of D2-like receptors robustly attenuated risk-taking, whereas drugs acting on D1-like receptors had no effect. Systemic amphetamine also reduced risk-taking, an effect which was attenuated by D2-like (but not D1-like) receptor blockade. Dopamine receptor mRNA expression was evaluated in a separate cohort of drug-naive rats characterized in the task. D1 mRNA expression in both nucleus accumbens shell and insular cortex was positively associated with risk-taking, while D2 mRNA expression in orbitofrontal and medial prefrontal cortex predicted risk preference in opposing nonlinear patterns. Additionally, lower levels of D2 mRNA in dorsal striatum were associated with greater risk-taking. These data strongly implicate dopamine signaling in prefrontal cortical-striatal circuitry in modulating decision-making processes involving integration of reward information with risks of adverse consequences.

Good things come to those who wait: attenuated discounting of delayed rewards in aged Fischer 344 rats.

The ability to make advantageous choices among outcomes that differ in magnitude, probability, and delay until their arrival is critical for optimal survival and well-being across the lifespan. Aged individuals are often characterized as less impulsive in their choices than their young adult counterparts, demonstrating an increased ability to forgo immediate in favor of delayed (and often more beneficial) rewards. Such "wisdom" is usually characterized as a consequence of learning and life experience. However, aging is also associated with prefrontal cortical dysfunction and concomitant impairments in advantageous choice behavior. Animal models afford the opportunity to isolate the effects of biological aging on decision-making from experiential factors. To model one critical component of decision-making, young adult and aged Fischer 344 rats were trained on a two-choice delay discounting task in which one choice provided immediate delivery of a small reward and the other provided a large reward delivered after a variable delay period. Whereas young adult rats showed a characteristic pattern of choice behavior (choosing the large reward at short delays and shifting preference to the small reward as delays increased), aged rats maintained a preference for the large reward at all delays (i.e., attenuated "discounting" of delayed rewards). This increased preference for the large reward in aged rats was not due to perceptual, motor, or motivational factors. The data strongly suggest that, independent of life experience, there are underlying neurobiological factors that contribute to age-related changes in decision-making, and particularly the ability to delay gratification.

Enhanced calcium buffering in F344 rat cholinergic basal forebrain neurons is associated with age-related cognitive impairment.

Alterations in neuronal Ca(2+) homeostasis are important determinants of age-related cognitive impairment. We examined the Ca(2+) influx, buffering, and electrophysiology of basal forebrain neurons in adult, middle-aged, and aged male F344 behaviorally assessed rats. Middle-aged and aged rats were characterized as cognitively impaired or unimpaired by water maze performance relative to young cohorts. Patch-clamp experiments were conducted on neurons acutely dissociated from medial septum/nucleus of the diagonal band with post hoc identification of phenotypic marker mRNA using single-cell RT-PCR. We measured whole cell calcium and barium currents and dissected these currents using pharmacological agents. We combined Ca(2+) current recording with Ca(2+)-sensitive ratiometric microfluorimetry to measure Ca(2+) buffering. Additionally, we sought changes in neuronal firing properties using current-clamp recording. There were no age- or cognition-related changes in the amplitudes or fractional compositions of the whole cell Ca(2+) channel currents. However, Ca(2+) buffering was significantly enhanced in cholinergic neurons from aged cognitively impaired rats. Moreover, increased Ca(2+) buffering was present in middle-aged rats that were not cognitively impaired. Firing properties were largely unchanged with age or cognitive status, except for an increase in the slow afterhyperpolarization in aged cholinergic neurons, independent of cognitive status. Furthermore, acutely dissociated basal forebrain neurons in which choline acetyltransferase mRNA was detected had the electrophysiological profiles of identified cholinergic neurons. We conclude that enhanced Ca(2+) buffering by cholinergic basal forebrain neurons may be important during aging.