Climbing the ladder from neuron to brain in Harold Atwood's laboratory.

Twenty years spent in one laboratory is sufficient to build a legacy of publications and a body of work to make an impact. However, the impact of our work was highest at the personal level, and time spent in Harold Atwood's laboratory was not a culmination of my career but rather a crucial path toward learning and maturing as a researcher. During that time, I experienced discoveries and lessons that shaped the next steps of my career. This article is written in gratitude for wonderful experiences and describes a few highlights that were especially memorable and influential.

γ-aminobutyric acid type A receptors that contain the δ subunit promote memory and neurogenesis in the dentate gyrus.

OBJECTIVE: Extrasynaptic γ-aminobutyric acid type A receptors that contain the δ subunit (δGABAA receptors) are highly expressed in the dentate gyrus (DG) subfield of the hippocampus, where they generate a tonic conductance that regulates neuronal activity. GABAA receptor-dependent signaling regulates memory and also facilitates postnatal neurogenesis in the adult DG; however, the role of the δGABAA receptors in these processes is unclear. Accordingly, we sought to determine whether δGABAA receptors regulate memory behaviors, as well as neurogenesis in the DG. METHODS: Memory and neurogenesis were studied in wild-type (WT) mice and transgenic mice that lacked δGABAA receptors (Gabrd(-/-)). To pharmacologically increase δGABAA receptor activity, mice were treated with the δGABAA receptor-preferring agonist 4,5,6,7-tetrahydroisoxazolo(5,4-c)pyridin-3-ol (THIP). Behavioral assays including recognition memory, contextual discrimination, and fear extinction were used. Neurogenesis was studied by measuring the proliferation, survival, migration, maturation, and dendritic complexity of adult-born neurons in the DG. RESULTS: Gabrd(-/-) mice exhibited impaired recognition memory and contextual discrimination relative to WT mice. Fear extinction was also impaired in Gabrd(-/-) mice, although the acquisition of fear memory was enhanced. Neurogenesis was disrupted in Gabrd(-/-) mice as the migration, maturation, and dendritic development of adult-born neurons were impaired. Long-term treatment with THIP facilitated learning and neurogenesis in WT but not Gabrd(-/-) mice. INTERPRETATION: δGABAA receptors promote the performance of certain DG-dependent memory behaviors and facilitate neurogenesis. Furthermore, δGABAA receptors can be pharmacologically targeted to enhance these processes.

Anatomical gradients of adult neurogenesis and activity: young neurons in the ventral dentate gyrus are activated by water maze training.

Hippocampal function varies in a subregion-specific fashion: spatial processing is thought to rely on the dorsal hippocampus, whereas anxiety-related behavior relies more on the ventral hippocampus. During development, neurogenesis in the dentate gyrus (DG) proceeds along ventral to dorsal as well as suprapyramidal to infrapyramidal gradients, but it is unclear whether regional differences in neurogenesis are maintained in adulthood. Moreover, it is unknown whether young neurons in the adult exhibit subregion-specific patterns of activation. We therefore examined the magnitude of neurogenesis and the activation of young and mature granule cells in DG subregions in adult rats that learned a spatial water maze task, swam with no platform, or were left untouched. We found that both adult neurogenesis and granule cell activation, as defined by c-fos expression in the granule cell population as a whole, were higher in the dorsal than the ventral DG. In contrast, c-fos expression in adult-born granule cells, identified by PSA-NCAM or location in the subgranular zone, occurred at a higher rate in the opposite subregion, the ventral DG. Interestingly, c-fos expression in the entire granule cell population was equivalent in water maze-trained rats and swim control rats, but was increased in the young granule cells only in the learning condition. These results provide new evidence that hippocampally-relevant experience activates young and mature neurons in different DG subregions and with different experiential specificity, and suggest that adult-born neurons may play a specific role in anxiety-related behavior or other nonspatial aspects of hippocampal function.

The effects of running and of inhibiting adult neurogenesis on learning and memory in rats.

The presence of ongoing adult neurogenesis within the highly plastic hippocampal circuitry poses questions as to the relevance of new neurons to learning and memory. Correlational and causal evidence suggests that some, but not all, hippocampal tasks involve the new neurons. The evidence with regard to spatial learning in the water maze, one of the most commonly used hippocampal tasks, is contradictory. In this study we examined the effects of irradiation-induced reduction in neurogenesis on spatial learning and another standard hippocampal task, contextual fear conditioning, in rats that experienced normal cage conditions or voluntary running. The results indicate that reduced neurogenesis had little effect on spatial learning but severely impaired contextual fear conditioning. It was suggested that compensatory mechanisms within the hippocampus may have contributed selectively to sparing of spatial function. Performance on the fear conditioning task was weakly related to enhanced neurogenesis or running. The results improve our understanding of the functional role of adult neurogenesis in behaving animals.

A model of hippocampal neurogenesis in memory and mood disorders.

The mounting evidence for neurogenesis in the adult hippocampus has fundamentally challenged the traditional view of brain development. The intense search for clues as to the functional significance of the new neurons has uncovered a surprising connection between neurogenesis and depression. In animal models of depression, neurogenesis is reduced, whereas many treatments for depression promote neurogenesis. We speculate on why the hippocampus, traditionally viewed as a memory structure, might be involved in mood disorders, and what specific role the new neurons might have in the pathogenesis of and recovery from depression. The proposed role of neurogenesis in contextual-memory formation predicts a specific pattern of cognitive deficits in depression and has important implications for treatment of this highly prevalent and debilitating disorder.

PAN-811 prevents chemotherapy-induced cognitive impairment and preserves neurogenesis in the hippocampus of adult rats.

Chemotherapy-induced cognitive impairment (CICI) occurs in a substantial proportion of treated cancer patients, with no drug currently available for its therapy. This study investigated whether PAN-811, a ribonucleotide reductase inhibitor, can reduce cognitive impairment and related suppression of neurogenesis following chemotherapy in an animal model. Young adult rats in Chemo and Chemo+PAN-811 groups received 3 intraperitoneal (i.p.) injections of methotrexate (MTX) and 5-fluorouracil (5-FU), and those in Saline and Saline+PAN-811 groups received equal volumes of physiological saline at 10-day intervals. PAN-811 in saline was delivered through i.p. injection, 10 min following each saline (Saline+PAN-811 group) or MTX/5-FU (Chemo+PAN-811 group) treatment, while equal volumes of saline were delivered to Saline and Chemo groups. Over Days 31-66, rats were administered tests of spatial memory, nonmatching-to-sample rule learning, and discrimination learning, which are sensitive to dysfunction in hippocampus, frontal lobe and striatum, respectively. On Day 97, neurogenesis was immnunohistochemically evaluated by counting doublecortin-positive (DCX+) cells in the dentate gyrus (DG). The results demonstrated that the Chemo group was impaired on the three cognitive tasks, but co-administration of PAN-811 significantly reduced all MTX/5-FU-induced cognitive impairments. In addition, MTX/5-FU reduced DCX+ cells to 67% of that in Saline control rats, an effect that was completely blocked by PAN-811 co-administration. Overall, we present the first evidence that PAN-811 protects cognitive functions and preserves neurogenesis from deleterious effects of MTX/5-FU. The current findings provide a basis for rapid clinical translation to determine the effect of PAN-811 on CICI in human.

Early Intervention with a Multi-Ingredient Dietary Supplement Improves Mood and Spatial Memory in a Triple Transgenic Mouse Model of Alzheimer's Disease.

The increasing global burden of Alzheimer's disease (AD) and failure of conventional treatments to stop neurodegeneration necessitates an alternative approach. Evidence of inflammation, mitochondrial dysfunction, and oxidative stress prior to the accumulation of amyloid-ß in the prodromal stage of AD (mild cognitive impairment; MCI) suggests that early interventions which counteract these features, such as dietary supplements, may ameliorate the onset of MCI-like behavioral symptoms. We administered a polyphenol-containing multiple ingredient dietary supplement (MDS), or vehicle, to both sexes of triple transgenic (3xTg-AD) mice and wildtype mice for 2 months from 2-4 months of age. We hypothesized that the MDS would preserve spatial learning, which is known to be impaired in untreated 3xTg-AD mice by 4 months of age. Behavioral phenotyping of animals was done at 1-2 and 3-4 months of age using a comprehensive battery of tests. As previously reported in males, both sexes of 3xTg-AD mice exhibited increased anxiety-like behavior at 1-2 months of age, prior to deficits in learning and memory, which did not appear until 3-4 months of age. The MDS did not reduce this anxiety or prevent impairments in novel object recognition (both sexes) or on the water maze probe trial (females only). Strikingly, the MDS specifically prevented 3xTg-AD mice (both sexes) from developing impairments (exhibited by untreated 3xTg-AD controls) in working memory and spatial learning. The MDS also increased sucrose preference, an indicator of hedonic tone. These data show that the MDS can prevent some, but not all, psychopathology in an AD model.

Neurobiological Mechanisms of Chemotherapy-induced Cognitive Impairment in a Transgenic Model of Breast Cancer.

Animal studies have reinforced clinical reports of cognitive impairment in cancer survivors following chemotherapy but, until now, all pre-clinical research in this area has been conducted on normal rodents. The present study investigated the effects of chemotherapy on cognition and underlying biological mechanisms in the FVB/N-Tg (MMTV-neu) 202 Mul/J mouse, a well-characterized transgenic model of breast cancer that has similarities to the tumorigenesis which occurs in humans. Tumor-bearing and control mice received three weekly injections of a combination of methotrexate + 5-fluorouracil, or an equal volume of saline. Different aspects of learning and memory were measured before and after treatment. The effects of tumor and chemotherapy on neurogenesis, neuro-inflammatory cytokine activity, and brain volume, as they relate to corresponding cognitive changes, were also measured. The toxic effects of chemotherapy extended to the cancerous model in which substantial cognitive impairment was also associated with the disease. Cognitive deficits were greatest in tumorigenic mice that received the anti-cancer drugs. Both tumor growth and chemotherapy caused significant changes in brain volume, including the hippocampus and frontal lobes, two structures that are directly implicated in cognitive tasks that were shown to be vulnerable. The level of hippocampal neurogenesis in adulthood was suppressed in chemotherapy-treated mice and associated with loss of hippocampus-controlled cognitive function. Dysregulation of cytokine activity was found in tumorigenic mice and associated with impaired cognitive performance. The results show that chemotherapy and tumor development independently contribute to cognitive deficits through different biological mechanisms.

Early-Age Running Enhances Activity of Adult-Born Dentate Granule Neurons Following Learning in Rats.

Cognitive reserve, the brain's capacity to draw on enriching experiences during youth, is believed to protect against memory loss associated with a decline in hippocampal function, as seen in normal aging and neurodegenerative disease. Adult neurogenesis has been suggested as a specific mechanism involved in cognitive (or neurogenic) reserve. The first objective of this study was to compare learning-related neuronal activity in adult-born versus developmentally born hippocampal neurons in juvenile male rats that had engaged in extensive running activity during early development or reared in a standard laboratory environment. The second objective was to investigate the long-term effect of exercise in rats on learning and memory of a contextual fear (CF) response later in adulthood. These aims address the important question as to whether exercise in early life is sufficient to build a reserve that protects against the process of cognitive aging. The results reveal a long-term effect of early running on adult-born dentate granule neurons and a special role for adult-born neurons in contextual memory, in a manner that is consistent with the neurogenic reserve hypothesis.

Environmental enrichment protects against cognitive impairment following chemotherapy in an animal model.

Clinical studies indicate that up to 70% of cancer patients who receive chemotherapy experience cognitive impairment. The present study investigated environmental enrichment as a protective factor against the adverse effects of anticancer drugs on cognitive and biological processes in an animal model. Adult rats were housed in group cages with environmental stimulation or in standard cages for 3 months, before receiving 3 weekly injections of methotrexate + 5-fluorouracil, or equal volumes of saline. Rats were then administered tests of learning and memory that are sensitive to hippocampal or frontal lobe dysfunction. The relationship between cognitive performance and hippocampal neurogenesis was examined through sensitive time-dependent measures of neuronal maturation. Chemotherapy-treated rats in the standard environment were impaired on tests of spatial memory, nonmatching-to-sample (NMTS) rule learning, and delayed-NMTS. Chemotherapy-treated rats in the enriched environment performed at or near normal levels. The performance of the chemotherapy groups on the hippocampus-sensitive, spatial memory and delayed-NMTS tests correlated with neurogenesis levels. The results show that environmental enrichment can reduce the risk of chemotherapy-induced cognitive impairment, in part by promoting neuronal differentiation and growth during cell maturation. As well, they point to the importance of lifestyle factors in treating or preventing adverse effects of anticancer drugs on cognitive function. (PsycINFO Database Record

Dynamics of neurogenesis in the dentate gyrus of adult rats.

Hippocampal neurogenesis declines steadily over the first year of life in the rodent, but the process persists into senescence despite a dramatic drop in the number of neurons it produces. At this point though, the survival and development patterns exhibited by new granule cells in the aging brain remain unclear in relation to patterns observed in the younger brain. The present study was carried out in order to obtain a direct quantitative comparison of hippocampal neurogenesis in juvenile and middle-aged rats with a high degree of temporal resolution, and to compare the survival and differentiation of the new cells over time. Thirty-eight-day-old and 12-month-old, male Sprague--Dawley rats were injected with 5-bromo-2'-deoxyuridine (BrdU) in order to label cells dividing in the dentate gyrus over a 24-h period, and immunohistochemical labeling was performed in order to record cell production and survival at eight different time points over the following two-month period. Using a marker of neuronally committed precursors and immature neurons (doublecortin; DCX), as well as a marker of mature neurons (calbindin d-28K; CaBP), the extent and timeline of neuronal differentiation, maturation, and migration of the new cells were also characterized. Results indicated that 12-month-old rats experienced a nearly 94% reduction in neurogenesis relative to juveniles, due almost entirely to a 92% drop in cell production. A largely preserved course of development and migration in the remaining newborn cells suggests treatments that enhance cell proliferation could be crucial in reversing the age-related decline in neurogenesis.

Memory loss in chemotherapy-treated rats is exacerbated in high-interference conditions and related to suppression of hippocampal neurogenesis.

Drugs used to treat cancer have neurotoxic effects that often produce memory loss and related cognitive deficits. In a test of the hypothesis that chemotherapy-induced cognitive impairment is related to a loss of inhibitory control, rats injected with a combination of methotrexate+5-fluouracil or equal volumes of saline, were administered a retroactive interference task in which memory for a learned discrimination problem was tested under conditions of high- and low-interference. The drugs had no effect on original learning or on re-learning the discrimination response when there was little interference, but the chemotherapy group was severely impaired in the hippocampus-sensitive, high-interference memory test. The impaired performance correlated significantly with reduced neurogenesis in the hippocampus. The failure to suppress interfering influences is consistent with a breakdown in pattern separation, a process that distinguishes and separates overlapping neural representations of experiences that have a high degree of similarity.

Organotypic slice cultures for studies of postnatal neurogenesis.

Here we describe a technique for studying hippocampal postnatal neurogenesis in the rodent brain using the organotypic slice culture technique. This method maintains the characteristic topographical morphology of the hippocampus while allowing direct application of pharmacological agents to the developing hippocampal dentate gyrus. Additionally, slice cultures can be maintained for up to 4 weeks and thus, allow one to study the maturation process of newborn granule neurons. Slice cultures allow for efficient pharmacological manipulation of hippocampal slices while excluding complex variables such as uncertainties related to the deep anatomic location of the hippocampus as well as the blood brain barrier. For these reasons, we sought to optimize organotypic slice cultures specifically for postnatal neurogenesis research.

Physical exercise prevents suppression of hippocampal neurogenesis and reduces cognitive impairment in chemotherapy-treated rats.

RATIONALE: Chemotherapy, used for the treatment of cancer, often produces cognitive impairment that has been related to suppression of neurogenesis. Physical exercise, which promotes neurogenesis, is known to improve cognitive function in neurologically challenged animals and humans. It is unknown whether exercise similarly protects against chemotherapy-induced cognitive impairment and whether recovery of neurogenesis is a critical factor. OBJECTIVE: The present study investigated the relationship between hippocampal neurogenesis and cognitive performance in chemotherapy-treated rats that engaged in different amounts of physical activity. METHODS: Groups of rats, housed individually in standard cages or in specially designed cages that allowed unlimited access to a running wheel, received three injections of the chemotherapeutic drugs methotrexate and 5-fluorouracil, or equal volumes of saline. They were then administered the following cognitive tests in a water maze: (1) spatial memory (SM), (2) cued memory, (3) non-matching to sample (NMTS) rule learning; (4) delayed NMTS (DNMTS). Hippocampal neurogenesis was quantified by counting doublecortin-expressing cells in the dentate gyrus. RESULTS: Chemotherapy administered to rats in standard cages resulted in a significant reduction in hippocampal neurogenesis and impaired performance on the SM, NMTS, and DNMTS tasks. In rats receiving chemotherapy and housed in exercise cages, neurogenesis was not suppressed and cognitive performance was similar to controls. CONCLUSIONS: Physical exercise can reduce cognitive deficits that result from chemotherapy and this effect is mediated, at least in part, by preventing suppression of drug-induced hippocampal neurogenesis. The results suggest benefits of exercise in preventing or treating cognitive impairment associated with chemotherapy.

Homeostatic regulation of adult hippocampal neurogenesis in aging rats: long-term effects of early exercise.

Adult neurogenesis is highly responsive to environmental and physiological factors. The majority of studies to date have examined short-term consequences of enhancing or blocking neurogenesis but long-term changes remain less well understood. Current evidence for age-related declines in neurogenesis warrant further investigation into these long-term changes. In this report we address the hypothesis that early life experience, such as a period of voluntary running in juvenile rats, can alter properties of adult neurogenesis for the remainder of the animal's life. The results indicate that the number of proliferating and differentiating neuronal precursors is not altered in runners beyond the initial weeks post-running, suggesting homeostatic regulation of these processes. However, the rate of neuronal maturation and survival during a 4 week period after cell division was enhanced up to 11 months of age (the end of the study period). This study is the first to show that a transient period of physical activity at a young age promotes changes in neurogenesis that persist over the long-term, which is important for our understanding of the modulation of neurogenesis by exercise with age. Functional integration of adult-born neurons within the hippocampus that resist homeostatic regulation with aging, rather than the absolute number of adult-born neurons, may be an essential feature of adult neurogenesis that promotes the maintenance of neural plasticity in old age.

Comorbid Aß toxicity and stroke: hippocampal atrophy, pathology, and cognitive deficit.

Numerous clinical and epidemiological reports indicate that patients with history of vascular illness such as stroke are more likely to develop dementia as the clinical manifestation of Alzheimer's disease. However, there are little data regarding the pathologic mechanisms that link vascular risk factors to the factors associated with dementia onset. We provide evidence that suggests intriguing detrimental interactions between stroke and ß-amyloid (Aß) toxicity in the hippocampus. Stroke was induced by unilateral striatal injection of endothelin-1, the potent vasoconstrictor. Aß toxicity was modeled by bilateral intracerebroventricular injections of the toxic fragment Aß. Gross morphologic changes in comorbid Aß and stroke rats were enlargement of the lateral ventricles with concomitant shrinkage of the hippocampus. The hippocampus displayed a series of synergistic biochemical alterations, including microgliosis, deposition of Aß precursor protein fragments, and cellular degeneration. In addition, there was bilateral induction of connexin43, reduced neuronal survival, and impaired dendritic development of adult-born immature neurons in the dentate gyrus of these rats compared with either rats alone. Behaviorally, there was impairment in the hippocampal-based discriminative fear-conditioning to context task indicating learning and memory deficit. These results suggest an insight into the relationship between hippocampal atrophy, pathology, and functional impairment. Our work not only highlights the exacerbated pathology that emerges when Aß toxicity and stroke occur comorbidly but also demonstrates that this comorbid rat model exhibits physiopathology that is highly characteristic of the human condition.

Adult hippocampal neurogenesis reduces memory interference in humans: opposing effects of aerobic exercise and depression.

Since the remarkable discovery of adult neurogenesis in the mammalian hippocampus, considerable effort has been devoted to unraveling the functional significance of these new neurons. Our group has proposed that a continual turnover of neurons in the DG could contribute to the development of event-unique memory traces that act to reduce interference between highly similar inputs. To test this theory, we implemented a recognition task containing some objects that were repeated across trials as well as some objects that were highly similar, but not identical, to ones previously observed. The similar objects, termed lures, overlap substantially with previously viewed stimuli, and thus, may require hippocampal neurogenesis in order to avoid catastrophic interference. Lifestyle factors such as aerobic exercise and stress have been shown to impact the local neurogenic microenvironment, leading to enhanced and reduced levels of DG neurogenesis, respectively. Accordingly, we hypothesized that healthy young adults who take part in a long-term aerobic exercise regime would demonstrate enhanced performance on the visual pattern separation task, specifically at correctly categorizing lures as "similar." Indeed, those who experienced a proportionally large change in fitness demonstrated a significantly greater improvement in their ability to correctly identify lure stimuli as "similar." Conversely, we expected that those who score high on depression scales, an indicator of chronic stress, would exhibit selective deficits at appropriately categorizing lures. As expected, those who scored high on the Beck Depression Inventory (BDI) were significantly worse than those with relatively lower BDI scores at correctly identifying lures as "similar," while performance on novel and repeated stimuli was identical. Taken together, our results support the hypothesis that adult-born neurons in the DG contribute to the orthogonalization of incoming information.

Adult neurogenesis. From circuits to models.

Our understanding of the hippocampus as a memory-encoding device is greatly helped by our knowledge of neuronal circuits and their plasticity. The trisynaptic hippocampal circuit carrying afferent input from the entorhinal cortex, controlled by a network of inhibitory interneurons and supplemented by modulatory subcortical inputs forms a platform for multiple forms of synaptic plastic mechanisms. Long-term potentiation of synaptic transmission in its various forms is an outstanding example of hippocampal ability to adapt to past neuronal activity. Adult neurogenesis is a profound plastic mechanism incorporating structural and functional changes that were previously thought to be present only in developing neural systems. These powerful forms of plasticity can mask experimental results by compensating for experimentally induced changes in the neurons or circuits. Circuit lesions have been one of the most common techniques in scientific investigations of the hippocampus. Although the effects of such lesions can be quite revealing and ground-breaking, in many cases the results are masked by compensatory mechanisms producing misleading results. This review will highlight such mechanisms and argue that the experimental results, in spite of their shortcomings, can be better understood when viewed in light of our knowledge of the neuronal circuitry, and with guidance by conceptual and computational models. Studies demonstrating a role of neurogenesis in pattern separation and memory interference are a good example of fruitful interaction between modeling and experimental approaches.

Adult hippocampal neurogenesis and memory interference.

Rats, subjected to low-dose irradiation that suppressed hippocampal neurogenesis, or a sham treatment, were administered a visual discrimination task under conditions of high, or low interference. Half of the rats engaged in running activity and the other half did not. In the non-runners, there was no effect of irradiation on learning, or remembering the discrimination response under low interference, but irradiation treatment increased their susceptibility to interference, resulting in loss of memory for the previously learned discrimination. Irradiated rats that engaged in running activity exhibited increased neuronal growth and protection from memory impairment. The results, which show that hippocampal cells generated in adulthood play a role in differentiating between conflicting, context-dependent memories, provide further evidence of the importance of neurogenesis in hippocampus-sensitive memory tasks. The results are consistent with computational models of hippocampal function that specify a central role for neurogenesis in the modulation of interfering influences during learning and memory.

The effects of chemotherapy on cognitive function in a mouse model: a prospective study.

PURPOSE: Clinical studies indicate that up to 70% of patients with cancer who receive chemotherapy experience cognitive impairment. The present study used a prospective longitudinal design to assess short- and long-term effects of commonly used anticancer drugs on cognitive performance in a mouse model. EXPERIMENTAL DESIGN: Normal mice received three weekly injections of a combination of methotrexate + 5-fluorouracil (CHEMO group) or an equal volume of saline (SAL group). Cognitive tests, measuring different aspects of learning and memory, were administered before treatment, immediately after treatment, and three months later. Structural MRI scanning was conducted at each stage of cognitive testing. RESULTS: The CHEMO group exhibited deficits on cognitive tasks acquired pretreatment [spatial memory, nonmatching-to-sample (NMTS) learning, and delayed NMTS], as well as impaired new learning on two tasks (conditional associative learning, discrimination learning) introduced posttreatment. Consistent with clinical evidence, cognitive deficits were pronounced on tests that are sensitive to hippocampal and frontal lobe dysfunction, but the CHEMO group's poor performance on the discrimination learning problem suggests that impairment is more widespread than previously thought. Cognitive deficits persisted for at least three months after treatment but some recovery was noted, particularly on tests thought to be under frontal lobe control. The MRI tests did not detect brain changes that could be attributed to treatment. CONCLUSIONS: Chemotherapeutic agents can have adverse effects on information acquired pretreatment as well as new learning and memory and, despite some recovery, impairment is long lasting.