Michelle M. Adams

Experience-dependent changes in cerebellar contributions to motor sequence learning

Studies in experimental animals and humans have stressed the role of the cerebellum in motor skill learning. Yet, the relative importance of the cerebellar cortex and deep nuclei, as well as the nature of the dynamic functional changes occurring between these and other motor-related structures during learning, remains in dispute. Using functional magnetic resonance imaging and a motor sequence learning paradigm in humans, we found evidence of an experience-dependent shift of activation from the cerebellar cortex to the dentate nucleus during early learning, and from a cerebellar–cortical to a striatal–cortical network with extended practice. The results indicate that intrinsic modulation within the cerebellum, in concert with activation of motor-related cortical regions, serves to set up a procedurally acquired sequence of movements that is then maintained elsewhere in the brain.

Circuit-specific alterations in hippocampal synaptophysin immunoreactivity predict spatial learning impairment in aged rats

The present study examined the long-standing concept that changes in hippocampal circuitry contribute to age-related learning impairment. Individual differences in spatial learning were documented in young and aged Long–Evans rats by using a hippocampal-dependent version of the Morris water maze. Postmortem analysis used a confocal laser-scanning microscopy method to quantify changes in immunofluorescence staining for the presynaptic vesicle glycoprotein, synaptophysin (SYN), in the principal relays of hippocampal circuitry. Comparisons based on chronological age alone failed to reveal a reliable difference in the intensity of SYN staining in any region that was examined. In contrast, aged subjects with spatial learning deficits displayed significant reductions in SYN immunoreactivity in CA3 lacunosum-moleculare (LM) relative to either young controls or age-matched rats with preserved learning. SYN intensity values for the latter groups were indistinguishable. In addition, individual differences in spatial learning capacity among the aged rats correlated with levels of SYN staining selectively in three regions: outer and middle portions of the dentate gyrus molecular layer and CA3-LM. The cross-sectional area of SYN labeling, by comparison, was not reliably affected in relation cognitive status. These findings are the first to demonstrate that a circuit-specific pattern of variability in the connectional organization of the hippocampus is coupled to individual differences in the cognitive outcome of normal aging. The regional specificity of these effects suggests that a decline in the fidelity of input to the hippocampus from the entorhinal cortex may play a critical role.

Different modes of hippocampal plasticity in response to estrogen in young and aged female rats

Estrogen regulates hippocampal dendritic spine density and synapse number in an N-methyl-d-aspartate (NMDA) receptor-dependent manner, and these effects may be of particular importance in the context of age-related changes in endocrine status. We investigated estrogens effects on axospinous synapse density and the synaptic distribution of the NMDA receptor subunit, NR1, within the context of aging. Although estrogen induced an increase in axospinous synapse density in young animals, it did not alter the synaptic representation of NR1, in that the amount of NR1 per synapse was equivalent across groups. Estrogen replacement in aged female rats failed to increase axospinous synapse density; however, estrogen up-regulated synaptic NR1 compared with aged animals with no estrogen. Therefore, the young and aged hippocampi react differently to estrogen replacement, with the aged animals unable to mount a plasticity response generating additional synapses, yet responsive to estrogen with respect to additional NMDA receptor content per synapse. These findings have important implications for estrogen replacement therapy in the context of aging.

Caloric restriction and age affect synaptic proteins in hippocampal CA3 and spatial learning ability.

Caloric restriction (CR) is a daily reduction of total caloric intake without a decrease in micronutrients or disproportionate reduction of any one dietary component. CR can increase lifespan reliably in a wide range of species and appears to counteract some aspects of the aging process throughout the body. The effects on the brain are less clear, but moderate CR seems to attenuate age-related cognitive decline. Thus, we determined the effects of age and CR on key synaptic proteins in the CA3 region of the hippocampus and whether these changes were correlated with differences in behavior on a hippocampal-dependent learning and memory task. We observed an overall, age-related decline in the NR1, N2A and N2B subunits of the N-methyl-d-aspartate (NMDA)-type and the GluR1 and GluR2 subunits of the alpha-amino-3-hydroxy-5-methyl-4-isoxazole proprionic acid (AMPA)-type ionotropic glutamate receptors. Interestingly, we found that CR initially lowers the glutamate receptor subunit levels as compared to young AL animals, and then stabilizes the levels across lifespan. Synaptophysin, a presynaptic vesicle protein, showed a similar pattern. We also found that both CR and ad libitum (AL) fed animals exhibited age-related cognitive decline on the Morris water maze task. However, AL animals declined between young and middle age, and between middle age and old, whereas CR rats only declined between young and middle age. Thus, the decrease in key synaptic proteins in CA3 and cognitive decline occurring across lifespan are stabilized by CR. This age-related decrease and CR-induced stabilization are likely to affect CA3 synaptic plasticity and, as a result, hippocampal function.

Visual Cortical Projections and Chemoarchitecture of Macaque Monkey Pulvinar

We investigated the patterns of projections from the pulvinar to visual areas V1, V2, V4, and MT, and their relationships to pulvinar subdivisions based on patterns of calbindin (CB) immunostaining and estimates of visual field maps (P1, P2 and P3). Multiple retrograde tracers were placed into V1, V2, V4, and/or MT in 11 adult macaque monkeys. The inferior pulvinar (PI) was subdivided into medial (PIM), posterior (PIP), central medial (PICM), and central lateral (PICL) regions, confirming earlier CB studies. The P1 map includes PICL and the ventromedial portion of the lateral pulvinar (PL), P2 is found in ventrolateral PL, and P3 includes PIP, PIM, and PICM. Projections to areas V1 and V2 were found to be overlapping in P1 and P2, but those from P2 to V2 were denser than those to V1. V2 also received light projections from PICM and, less reliably, from PIM. Neurons projecting to V4 and MT were more abundant than those projecting to V1 and V2. Those projecting to V4 were observed in P1, densely in P2, and also in PICM and PIP of P3. Those projecting to MT were found in P1– P3, with the heaviest projection from P3. Projections from P3 to MT and V4 were mainly interdigitated, with the densest to MT arising from PIM and the densest to V4 arising from PIP and PICM. Because the calbindin‐rich and ‐poor regions of P3 corresponded to differential patterns of cortical connectivity, the results suggest that CB may further delineate functional subdivisions in the pulvinar. J. Comp. Neurol. 419:377–393, 2000.

Estrogen modulates synaptic N-methyl-D-aspartate receptor subunit distribution in the aged hippocampus.

Estrogen interacts with N‐methyl‐D‐aspartate (NMDA) receptors to regulate multiple aspects of morphological and functional plasticity. In hippocampus, estrogen increases both dendritic spine density and synapse number, and NMDA antagonists block these effects. Thus, estrogen‐mediated hippocampal plasticity may be of particular importance in the context of age‐related changes in endocrine status and cognitive performance. NR1 levels per synapse are increased in CA1 by estrogen in aged rats but not young rats, although no information is available on estrogen‐induced synaptic alterations in other NMDA receptor subunits that might impact function. Therefore, the present study was designed to investigate the effect of estrogen on the synaptic and subsynaptic distributions of the NMDA receptor subunits, NR2A and NR2B in CA1 pyramidal cells, within the context of aging. Our results demonstrated that the overall synaptic levels of NR2A and NR2B are similar in young and aged female rats, regardless of estrogen treatment. However, in the aged CA1, estrogen restores NR2B levels back to young levels in the lateral portions of the active synaptic zone. Thus, estrogen may impact the mobility of NMDA receptors across the synapse and, in the process, restore a more youthful synaptic profile. These findings have important implications for the mechanism of estrogen‐induced alterations in NMDA receptor‐mediated processes, particularly in the context of aging. J. Comp. Neurol. 474:419–426, 2004.

Neurofilament protein is differentially distributed in subpopulations of corticocortical projection neurons in the macaque monkey visual pathways.

Previous studies of the primate cerebral cortex have shown that neurofilament protein is present in pyramidal neuron subpopulations displaying specific regional and laminar distribution patterns. In order to characterize further the neurochemical phenotype of the neurons furnishing feedforward and feedback pathways in the visual cortex of the macaque monkey, we performed an analysis of the distribution of neurofilament protein in corticocortical projection neurons in areas V1, V2, V3, V3A, V4, and MT. Injections of the retrogradely transported dyes Fast Blue and Diamidino Yellow were placed within areas V4 and MT, or in areas V1 and V2, in 14 adult rhesus monkeys, and the brains of these animals were processed for immunohistochemistry with an antibody to nonphosphorylated epitopes of the medium and heavy molecular weight subunits of the neurofilament protein. Overall, there was a higher proportion of neurons projecting from areas V1, V2, V3, and V3A to area MT that were neurofilament protein‐immunoreactive (57–100%), than to area V4 (25–36%). In contrast, feedback projections from areas MT, V4, and V3 exhibited a more consistent proportion of neurofilament protein‐containing neurons (70–80%), regardless of their target areas (V1 or V2). In addition, the vast majority of feedback neurons projecting to areas V1 and V2 were located in layers V and VI in areas V4 and MT, while they were observed in both supragranular and infragranular layers in area V3. The laminar distribution of feedforward projecting neurons was heterogeneous. In area V1, Meynert and layer IVB cells were found to project to area MT, while neurons projecting to area V4 were particularly dense in layer III within the foveal representation. In area V2, almost all neurons projecting to areas MT or V4 were located in layer III, whereas they were found in both layers II–III and V–VI in areas V3 and V3A. These results suggest that neurofilament protein identifies particular subpopulations of corticocortically projecting neurons with distinct regional and laminar distribution in the monkey visual system. It is possible that the preferential distribution of neurofilament protein within feedforward connections to area MT and all feedback projections is related to other distinctive properties of these corticocortical projection neurons.

Hippocampal dependent learning ability correlates with N-methyl-D-aspartate (NMDA) receptor levels in CA3 neurons of young and aged rats

Hippocampal N‐methyl‐D‐Aspartate (NMDA) receptors mediate mechanisms of cellular plasticity critical for spatial learning in rats. The present study examined the relationship between spatial learning and NMDA receptor expression in discrete neuronal populations, as well as the degree to which putative age‐related changes in NMDA receptors are coupled to the effects of normal aging on spatial learning. Young and aged Long‐Evans rats were tested in a Morris water maze task that depends on the integrity of the hippocampus. Levels of NR1, the obligatory subunit for a functional NMDA receptor, were subsequently quantified both biochemically by Western blot in whole homogenized hippocampus, and immunocytochemically by using a high‐resolution confocal laser scanning microscopy method. The latter approach allowed comprehensive, regional analysis of discrete elements of excitatory hippocampal circuitry. Neither method revealed global changes, nor were there region‐specific differences in hippocampal NR1 levels between young and aged animals. However, across all subjects, individual differences in spatial learning ability correlated with NR1 immunofluorescence levels selectively in CA3 neurons of the hippocampus. Parallel confocal microscopic analysis of the GluR2 subunit of the alpha‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazole proprionic acid (AMPA) receptor failed to reveal reliable differences as a function of age or spatial learning ability. This analysis linking age, performance, and NR1 levels demonstrates that although dendritic NR1 is generally preserved in the aged rat hippocampus, levels of this receptor subunit in selective elements of hippocampal circuitry are linked to spatial learning. These findings suggest that NMDA receptor abundance in CA3 bears a critical relationship to learning mediated by the hippocampus throughout the life span. J. Comp. Neurol. 432:230–243, 2001.

Caloric restriction eliminates the aging-related decline in NMDA and AMPA receptor subunits in the rat hippocampus and induces homeostasis

Caloric restriction (CR) extends life span and ameliorates the aging-related decline in hippocampal-dependent cognitive function. In the present study, we compared subunit levels of NMDA and AMPA types of the glutamate receptor and quantified total synapses and multiple spine bouton (MSB) synapses in hippocampal CA1 from young (10 months), middle-aged (18 months), and old (29 months) Fischer 344xBrown Norway rats that were ad libitum (AL) fed or caloric restricted (CR) from 4 months of age. Each of these parameters has been reported to be a potential contributor to hippocampal function. Western blot analysis revealed that NMDA and AMPA receptor subunits in AL animals decrease between young and middle age to levels that are present at old age. Interestingly, young CR animals have significantly lower levels of glutamate receptor subunits than young AL animals and those lower levels are maintained across life span. In contrast, stereological quantification indicated that total synapses and MSB synapses are stable across life span in both AL and CR rats. These results indicate significant aging-related losses of hippocampal glutamate receptor subunits in AL rats that are consistent with altered synaptic function. CR eliminates that aging-related decline by inducing stable NMDA and AMPA receptor subunit levels.

Significance of N-methyl-D-aspartate (NMDA) receptor-mediated signaling in human keratinocytes

Increasing data suggest that glutamate might act as a cell‐signaling molecule in non‐neuronal tissues such as the skin. Here we demonstrate the presence of functional N‐methyl‐d‐aspartate (NMDA)‐type glutamate receptors in human keratinocytes. NMDA receptor expression strongly reflects the degree of cell‐to‐cell contact. Wounding polarizes the expression of NMDA receptors in keratinocytes involved in re‐epithelialization, and the process of re‐epithelialization is inhibited by NMDA receptor activation. We also demonstrate that squamous cell carcinomas lack NMDA receptors. Our data suggest that Ca2+ entry through NMDA receptors influences the cycle of keratinocyte proliferation, differentiation, and migration during epithelialization. Moreover, NMDA receptor activation might play a role in contact‐mediated inhibition of growth, a process that is absent during neoplastic pathology. This receptor may serve as a pharmacological target for modulating keratinocyte behavior and treating cutaneous disorders.