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1.  Pepsin Pretreatment Allows Collagen IV Immunostaining of Blood Vessels in Adult Mouse Brain 
Journal of neuroscience methods  2007;163(1):76-82.
While the brain vasculature can be imaged with many methods, immunohistochemistry has distinct advantages due to its simplicity and applicability to archival tissue. However, immunohistochemical staining of the murine brain vasculature in aldehyde fixed tissue has proven elusive and inconsistent using current protocols. Here we investigated whether antigen retrieval methods could improve vascular staining in the adult mouse brain. We found that pepsin digestion prior to immunostaining unmasked widespread collagen IV staining of the cerebrovasculature in the adult mouse brain. Pepsin treatment also unmasked widespread vascular staining with laminin, but only marginally improved isolectin B4 staining and did not enhance vascular staining with fibronectin, perlecan or CD146. Collagen IV immunoperoxidase staining was easily combined with cresyl violet counterstaining making it suitable for stereological analyses of both vascular and neuronal parameters in the same tissue section. This method should be widely applicable for labeling the brain vasculature of the mouse in aldehyde fixed tissue from both normal and pathological states.
PMCID: PMC1931483  PMID: 17403541
adult; antigen retrieval; blood vessels; brain; collagen IV; immunohistochemistry; mouse; pepsin
2.  Interactive Effects of Age and Estrogen on Cortical Neurons: Implications for Cognitive Aging 
Neuroscience  2011;191:148-158.
In the past few decades it has become clear that estrogen signaling plays a much larger role in modulating the cognitive centers of the brain than previously thought possible. We have developed a nonhuman primate (NHP) model to investigate the relationships between estradiol (E) and cognitive aging. Our studies of cyclical E treatment in ovariectomized (OVX) young and aged rhesus monkeys have revealed compelling cognitive and synaptic effects of E in the context of aging. Delayed response (DR), a task that is particularly dependent on integrity of dorsolateral prefrontal cortex (dlPFC) area 46 revealed the following: 1) that young OVX rhesus monkeys perform equally well whether treated with E or vehicle (V), and 2) that aged OVX animals given E perform as well as young adults with or without E, whereas OVX V-treated aged animals display significant DR impairment. We have analyzed the structure of layer III pyramidal cells in area 46 in these same monkeys. We found both age and treatment effects on these neurons that are consistent with behavioral data. Briefly, reconstructions of pyramidal neurons in area 46 from these monkeys showed that cyclical E increased the density of small, thin spines in both young and aged monkeys. However, this effect of E was against a background of age-related loss of small, thin spines, leaving aged V-treated monkeys with a particularly low density of these highly plastic spines and vulnerable to cognitive decline. Our current interpretation is that E not only plays a critically important role in maintaining spine number, but also enables synaptic plasticity through a cyclical increase in small highly plastic spines that may be stabilized in the context of learning. Interestingly, recent studies demonstrate that chronic E is less effective at inducing spinogenesis than cyclical E. We have begun to link certain molecular attributes of excitatory synapses in area 46 to E effects and cognitive performance in these monkeys. Given the importance of synaptic estrogen receptor α (ER-α) in rat hippocampus, we focused our initial studies on synaptic ER-α in area 46. Three key findings have emerged from these studies: 1) synaptic ER-α is present in axospinous synapses in area 46; 2) it is stable across treatment and age groups (which is not the case in rat hippocampus); and 3) the abundance and distribution of synaptic ER-α is a key correlate of individual variation in cognitive performance in certain age and treatment groups. These findings have important implications for the design of hormone treatment strategies for both surgically and naturally menopausal women.
PMCID: PMC3166405  PMID: 21664255
Prefrontal cortex; estrogen; aging; primate; cognition; hormone replacement therapy
3.  The distribution of NMDA and AMPA receptor subunits at thalamo-amygdala dendritic spines 
Brain research  2007;1134(1):87-94.
Synapses onto dendritic spines in the lateral amygdala formed by afferents from the auditory thalamus represent a site of plasticity in Pavlovian fear conditioning. Previous work has demonstrated that thalamic afferents synapse onto LA spines expressing glutamate receptor (GluR) subunits, but the GluR subunit distribution at the synapse and within the cytoplasm has not been characterized. Therefore, we performed a quantitative analysis for ∝-amino-3-hydroxy-5-methyl-4-isoxazole proprionate (AMPA) receptor subunits GluR2 and GluR3 and N-methyl-D-aspartate (NMDA) receptor subunits NR1 and NR2B by combining anterograde labeling of thalamo-amygdala afferents with postembedding immunoelectron microscopy for the GluRs in adult rats. A high percentage of thalamo-amygdala spines was immunoreactive for GluR2 (80%), GluR3 (83%), and NR1 (83%), while a smaller proportion of spines expressed NR2B (59%). To compare across the various subunits, the cytoplasmic to synaptic ratios of GluRs were measured within thalamo-amygdala spines. Analyses revealed that the cytoplasmic pool of GluR2 receptors was twice as large compared to the GluR3, NR1 and NR2B subunits. Our data also show that in adult brain, the NR2B subunit is expressed in the majority of in thalamo-amygdala spines and that within these spines, the various GluRs are differentially distributed between synaptic and non-synaptic sites. The prevalence of the NR2B subunit in thalamo-amygdala spines provides morphological evidence supporting its role in the fear conditioning circuit while the differential distribution of the GluR subtypes may reflect distinct roles for their involvement in this circuitry and synaptic plasticity.
PMCID: PMC2359729  PMID: 17207780
GluR2; GluR3; excitatory amino acids; immunogold; NR1; NR2B; postembedding; immunohistochemistry; tracing; electron microscopy

Results 1-3 (3)