Heather McKay

Estrogen Alters Spine Number and Morphology in Prefrontal Cortex of Aged Female Rhesus Monkeys

Long-term cyclic treatment with 17β-estradiol reverses age-related impairment in ovariectomized rhesus monkeys on a test of cognitive function mediated by the prefrontal cortex (PFC). Here, we examined potential neurobiological substrates of this effect using intracellular loading and morphometric analyses to test the possibility that the cognitive benefits of hormone treatment are associated with structural plasticity in layer III pyramidal cells in PFC area 46. 17β-Estradiol did not affect several parameters such as total dendritic length and branching. In contrast, 17β-estradiol administration increased apical and basal dendritic spine density, and induced a shift toward smaller spines, a response linked to increased spine motility, NMDA receptor-mediated activity, and learning. These results document that, although the aged primate PFC is vulnerable in the absence of factors such as circulating estrogens, it remains responsive to long-term cyclic 17β-estradiol treatment, and that increased dendritic spine density and altered spine morphology may contribute to the cognitive benefits of such treatment.

Estrogen increases the number of spinophilin-immunoreactive spines in the hippocampus of young and aged female rhesus monkeys.

It is well documented that estrogen increases dendritic spine density in CA1 pyramidal cells of young female rats. However, this effect is attenuated in aged rats. We report here a quantitative analysis of estrogen effects on hippocampal spine number as visualized with antispinophilin in young (6–8 years old) and aged (19–23 years old) female rhesus monkeys, a species with a pattern of female endocrine senescence comparable to that of humans. Monkeys were ovariectomized and administered either vehicle or estradiol cypionate 3 months postovariectomy, followed by an additional dose 3 weeks later, with perfusion 24 hours after the last estrogen treatment. Immunolocalization of spinophilin, a spine‐associated protein, was used for quantitative stereologic analyses of total spinophilin‐immunoreactive spine numbers in CA1 stratum radiatum and the inner and outer molecular layers of dentate gyrus. In both young and aged female monkeys, the estrogen‐treated groups had an increase in spinophilin‐immunoreactive spines (37% in young, P < .005; 35% in aged, P < .05) compared with the untreated groups that amounted to more than 1 billion additional immunoreactive spines. The young group also showed a trend toward an estrogen‐induced increase in immunoreactive spines in the dentate gyrus outer molecular layer, but this effect was not statistically significant (P = .097). We conclude that spine number in the rhesus monkey hippocampus is highly responsive to estrogen, yet, unlike the female rat, aged female rhesus monkeys retain the capacity for spine induction in response to estrogen. These data have important implications for cognitive effects of estrogen replacement in postmenopausal women and demonstrate that an estrogen replacement protocol that mimics normal physiological cycles with timed, intermittent peaks can have profound neurobiological effects. J. Comp. Neurol. 465:540–550, 2003.

Endogenous Neurogenesis Replaces Oligodendrocytes and Astrocytes after Primate Spinal Cord Injury

Neurogenesis has been described in various regions of the CNS throughout life. We examined the extent of natural cell division and replacement from 7 weeks to 7 months after cervical spinal cord injury in four adult rhesus monkeys. Bromodeoxyuridine (BrdU) injections revealed an increase of >80-fold in the number of newly divided cells in the primate spinal cord after injury, with an average of 725,000 BrdU-labeled cells identified per monkey in the immediate injury zone. By 7 months after injury, 15% of these new cells expressed mature markers of oligodendrocytes and 12% expressed mature astrocytic markers. Newly born oligodendrocytes were present in zones of injury-induced demyelination and appeared to ensheath or remyelinate host axons. Thus, cell replacement is an extensive natural compensatory response to injury in the primate spinal cord that contributes to neural repair and is a potential target for therapeutic enhancement.

Bilateral Corticospinal Projections Arise from Each Motor Cortex in the Macaque Monkey: A Quantitative Study

The corticospinal projection is considered to influence fine motor function through nearly exclusively contralateral projections from the cortex in primates. However, unilateral lesions to this system in various species are frequently followed by significant functional improvement, raising the possibility that bilateral projections of this pathway may exist or emerge after injury. To examine the detailed anatomy and projections of the corticospinal motor neurons in rhesus monkeys (n = 4), we injected the high‐resolution anterograde tracer biotinylated dextran amine (BDA) into 126 sites centered about the right lower extremity (LE) primary motor cortex. Projection and termination patterns were quantified at lumbar levels L1, L4, and L7 and mapped by using serial‐section reconstructions. Notably, a mean of 10.1 ± 0.6% (± SEM) of corticospinal tract (CST) axons descended in the lateral CST ipsilateral to the cortical BDA injection, and 87.9 ± 1.0% of total CST axons projected in the contralateral lateral CST. The ipsilateral ventral CST contained only 1.0 ± 0% of all projecting CST axons, whereas the contralateral ventral CST contained 0.3 ± 0.2% of all axons. In addition, a minor dorsal column CST projection was identified. Measurement of BDA‐labeled terminals in the spinal cord gray matter revealed that 11.2 ± 2.2% of CST axons terminated ipsilateral to the side of cortical injection, and the remainder terminated contralaterally. As previously reported, most CST axons terminated in spinal cord laminae V–VIII, as well as the laterodorsal motoneuronal group of lamina IX (which innervates distal extremity muscles). Notably, many CST axons crossed the spinal cord midline (mean 19.9 ± 4.9 axons per 40‐μm‐thick section). Detailed single‐axon reconstructions revealed that most ipsilaterally projecting lateral CST axons terminated in ipsilateral gray matter. Notably, we found that the bouton‐like swellings of many ipsilateral CST axons descending in the dorsolateral tract were located within Rexeds lamina IX, in close proximity to motoneuronal somata. Thus, bilateral projections of corticospinal axons originating from a single motor cortex could contribute to bilateral control of spinal motor neurons and to the highly evolved degree of fine motor control in primates. Furthermore, bilateral CST projections from a single motor cortex could represent a potential source of plasticity after injury, as well as a target of therapeutic effort in neural regeneration strategies. J. Comp. Neurol. 473:147–161, 2004.

Memory Impairment in Aged Primates Is Associated with Focal Death of Cortical Neurons and Atrophy of Subcortical Neurons

Mechanisms of cognitive decline with aging remain primarily unknown. We determined whether localized cell loss occurred in brain regions associated with age-related cognitive decline in primates. On a task requiring the prefrontal cortex, aged monkeys were impaired in maintaining representations in working memory. Stereological quantification in area 8A, a prefrontal region associated with working memory, demonstrated a significant 32 ± 11% reduction in the number of Nissl-stained neurons compared with young monkeys. Furthermore, the number of immunolabeled cholinergic neurons projecting to this region of cortex from the nucleus basalis was also reduced by 50 ± 6%. In contrast, neuronal number was strikingly preserved in an adjoining prefrontal cortical region also associated with working memory, area 46, and in the component of the nucleus basalis projecting to this region. These findings demonstrate extensive but highly localized loss of neocortical neurons in aged, cognitively impaired monkeys that likely contributes to cognitive decline. Cell degeneration, when present, extends transneuronally.

Spontaneous and augmented growth of axons in the primate spinal cord: effects of local injury and nerve growth factor-secreting cell grafts.

Little is known about molecular and cellular responses to spinal cord injury in primates. In this study, the normal milieu of the primate spinal cord was disturbed by multiple needle penetrations and cell injections in the mid‐thoracic spinal cord; subsequent effects on local axons and expression of extracellular matrix (ECM) molecules were examined, together with effects of cellular delivery of nerve growth factor (NGF) to the injured region. Four adult rhesus monkeys each received injections of two grafts of autologous fibroblasts genetically modified to secrete human NGF, and, in control injection sites, two separate grafts of autologous fibroblasts transduced to express the reporter gene, β‐galactosidase. Three months later, Schwann cells extensively infiltrated the region of localized injury and penetrated both NGF and control fibroblast grafts. Marked upregulation of several ECM molecules occurred, including chondroitin and heparan sulfate proteoglycans and type IV collagen, in or adjacent to all injection sites. Schwann cells were an apparent source of some ECM expression. Spinal cord sensory axons and putative coerulospinal axons extended into both graft types, but they penetrated NGF grafts to a significantly greater extent. Many of these axons expressed the cell adhesion molecule L1. Thus, extensive cellular and molecular changes occur at sites of localized primate spinal cord injury and grafting, attributable in part to migrating Schwann cells, and are accompanied by spontaneous axonal plasticity. These molecular and cellular events closely resemble those observed in the rodent spinal cord after injury. Furthermore, as in rodent studies, cellular delivery of a trophic factor significantly augments axonal plasticity in the primate spinal cord. J. Comp. Neurol. 449:88–101, 2002.

Effects of estrogen replacement therapy on cholinergic basal forebrain neurons and cortical cholinergic innervation in young and aged ovariectomized rhesus monkeys

Estrogen modulates the function of cholinergic basal forebrain neurons in aged female rats. The present study tested the hypothesis that estrogen enhances the phenotype of cholinergic basal forebrain neurons and their cortical cholinergic innervation in young adult and aged ovariectomized rhesus monkeys. Sixteen monkeys (9 young and 7 aged) received two injections of estradiol cypionate or vehicle separated by 3 weeks. All monkeys were killed 1 day after the last injection. Quantitative immunofluorescence in the vertical limb of the diagonal band (VLDB) revealed enhanced optical density for choline acetyltransferase (ChAT) in both young and aged monkeys treated with estrogen. In contrast, optical density for low‐affinity p75 neurotrophin receptor immunoreactivity in the VLDB did not change after estrogen treatment in either aged or young animals. Quantitative immunofluorescence for either ChAT or the low‐affinity p75 neurotrophin receptor in the nucleus basalis Meynert failed to reveal differences between vehicle and estrogen treatment in either age group. Quantitative estimates of acetylcholinesterase (AChE) fiber density revealed that estrogen‐treated aged monkeys but not their younger counterparts had decreased numbers of AChE‐positive fibers in layer II of frontal, insular, and cingulate cortices. These data indicate that estrogen administered in a manner simulating natural hormonal cyclicity produces modest age‐specific chemical phenotypic and regional changes in select neuronal subfields of the cholinergic basal forebrain and their cortical projection sites in nonhuman primates. J. Comp. Neurol. 472:193–207, 2004.