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author:("Aoki, chile")
1.  Excitatory Synapses on Dendritic Shafts of the Amygdala Exhibit Elevated Levels of GABAA Receptor α4 Subunits Following the Induction of Activity-Based Anorexia 
Synapse (New York, N.Y.)  2013;68(1):1-15.
Anorexia nervosa (AN) is an eating disorder characterized by self-imposed severe starvation, excessive exercise, and anxiety. The onset of AN is most often at puberty, suggesting that gonadal hormonal fluctuations may contribute to AN vulnerability. Activity-based anorexia (ABA) is an animal model that reproduces some of the behavioral phenotypes of AN, including the paradoxical increase in voluntary exercise following food restriction. The basal amygdala as well as the GABAergic system regulate trait anxiety. We therefore examined the subcellular distribution of GABA receptors (GABARs) in the basal amygdala of female pubertal rats and specifically of their α4 subunits, because expression of α4-containing GABARs is regulated by gonadal hormone fluctuations. Moreover, because these GABARs reduce neuronal excitability through shunting of EPSPs, we quantified the frequency of occurrence of these GABARs adjacent to excitatory synapses. Electron microscopic immunoctychemistry revealed no change in the frequency of association of α4 subunits with excitatory synapses on dendritic spines, whether in the anterior (Bregma −2.8 mm) or caudal (Bregma −3.8 mm) portion of the basal amygdala. Sholl analysis of golgi-stained neurons also revealed no change in the extent of dendritic branching by these densely spiny, pyramidal-like neurons. However, there was an increase of membranous α4 subunits near excitatory synapses on dendritic shafts, specifically in the amygdala, and this was accompanied by a rise of α4 subunits intracellularly. Because most dendritic shafts exhibiting excitatory synapses are GABAergic interneurons, the results predict disinhibition, which would increase excitability of the amygdaloid network, in turn augmenting ABA animals’ anxiety.
PMCID: PMC4111138  PMID: 23766101
exercise; food restriction; anorexia nervosa; anxiety; hyperactivity; disinhibition
2.  Decreased expression of GLT-1 in the R6/2 model of Huntington’s disease does not worsen disease progression 
The European journal of neuroscience  2013;38(3):2477-2490.
Excitotoxicity is thought to be important in the pathogenesis of Huntington’s disease (HD). Glutamate is the predominant excitatory neurotransmitter in the brain and excess activation of glutamate receptors can cause neuronal dysfunction and death. Glutamate transporters regulate the extracellular concentration of glutamate. GLT-1 is the most abundant known glutamate transporter and accounts for most of the glutamate transport in the brain. Administration of ceftriaxone, an antibiotic that increases the functional expression of GLT-1, can improve the behavioral phenotype of the R6/2 mouse model of HD. To test the hypothesis that GLT-1 expression critically affects the HD disease process, we generated a novel mouse model that is heterozygous for the null allele of GLT-1 and carries the R6/2 transgene (double mutation). We demonstrated that the protein expression of total GLT-1, as well as two of its isoforms, are decreased within the cortex and striatum of 12-week-old R6/2 mice and that the expression of EAAC1 was decreased in the striatum. Protein expression of GLT-1 was further decreased in the cortex and striatum of the double mutation mice compared to the R6/2 mice at 11 weeks of age. However, the effects of the R6/2 transgene on weight loss, accelerating rotarod, climbing and paw-clasping were not exacerbated in these double mutants. Na+-dependent glutamate uptake into synapatosomes isolated from the striatum and cortex of 11-week-old R6/2 mice was unchanged compared to controls. These results suggest that changes in GLT-1 expression or function per se are unlikely to potentiate or ameliorate the progression of HD.
PMCID: PMC3735860  PMID: 23586612
Glutamate transporter; R6/2; synaptosomes; Na+-dependent glutamate uptake; EM-ICC
3.  Presenilin Conditional Double Knockout Mice Exhibit Decreases in Drebrin A at Hippocampal CA1 Synapses 
Synapse (New York, N.Y.)  2012;66(10):870-879.
Presenilin conditional double knockout (PScDKO) mice have been used as animal models to study the development of Alzheimer’s disease (AD) phenotypes. Studies to date indicate that these animals exhibit memory dysfunction and decreased synaptic plasticity before the onset of neurodegeneration. Therefore, the current study sought to examine how the loss of presenilin expression leads to these defects. Drebrin A, a neuron-specific actin-binding protein, has been shown to play an important role in the activity-dependent redistribution of the NMDA type of glutamate receptors at the synapse which, in turn, is a critical step for enabling synaptic plasticity. It is hypothesized that defects in the activity dependent redistribution of NMDA receptors in PScDKO mice may be due to loss of drebrin A. In this study, electron microscopic immunocytochemistry (EM-ICC) was used to quantify and locate drebrin A in the CA1 field of the hippocampus of PScDKO mice. The high resolution of EM-ICC allowed for differentiation between drebrin A at the synapse and at nonsynaptic sites, the latter of which would reflect the protein’s role in regulating the reserve or degradative pool of NMDA receptors. The results here demonstrate that loss of function of presenilin in mice leads to a decrease in immunoreactivity for drebrin A at both synaptic (54% decrease, P < 0.01) and nonsynaptic areas (40% decrease, P < 0.01) and overall (44% decrease, P < 0.01). The reduction of drebrin A in both synaptic and non-synaptic locations of the spine may implicate impairment in glutamate receptor trafficking to and from the postsynaptic plasma membrane. In addition, because of reduced drebrin A at nonsynaptic sites, the regulation of the reserve and degradative pools of glutamate receptors may also be impaired, leading to further synaptic dysfunction. Therefore, this study provides evidence to the theory that drebrin A has a causal role in compromising activity-dependent glutamate receptor trafficking in PScDKO mice.
PMCID: PMC4099411  PMID: 22715045
electron microscopy; immunocytochemistry; drebrin A; Alzheimer’s disease; CA1; hippocampus; mouse; animal model
4.  Adolescent female C57BL/6 mice with vulnerability to activity-based anorexia exhibit weak inhibitory input onto hippocampal CA1 pyramidal cells 
Neuroscience  2013;241:250-267.
Anorexia nervosa (AN) is an eating disorder characterized by self-imposed severe starvation and often linked with excessive exercise. Activity-based anorexia (ABA) is an animal model that reproduces some of the behavioral phenotypes of AN, including the paradoxical increase in voluntary exercise following food restriction (FR). Although certain rodents have been used successfully in this animal model, C57BL/6 mice are reported to be less susceptible to ABA. We re-examined the possibility that female C57BL/6 mice might exhibit ABA vulnerability during adolescence, the developmental stage/sex among the human population with particularly high AN vulnerability. After introducing the running wheel to the cage for three days, ABA was induced by restricting food access to 1 hour per day (ABA1, N=13) or 2 hours per day (ABA2, N=10). All 23 exhibited increased voluntary wheel running (p<0.005) and perturbed circadian rhythm within two days. Only one out of five survived ABA1 for three days, while ten out of ten survived ABA2 for three days and could subsequently restore their body weight and circadian rhythm. Exposure of recovered animals to a second ABA2 induction revealed a large range of vulnerability, even within littermates. To look for the cellular substrate of differences in vulnerability, we began by examining synaptic patterns in the hippocampus, a brain region that regulates anxiety as well as plasticity throughout life. Quantitative EM analysis revealed that CA1 pyramidal cells of animals vulnerable to the second ABA2 exhibit less GABAergic innervation on cell bodies and dendrites, relative to the animals resilient to the second ABA (p<0.001) or controls (p<0.05). These findings reveal that C57BL/6J adolescent females can be used to capture brain changes underlying ABA vulnerability, and that GABAergic innervation of hippocampal pyramidal neurons is one important cellular substrate to consider for understanding the progression of and resilience to AN.
PMCID: PMC3954564  PMID: 23523748
anorexia nervosa; eating disorder; exercise; relapse; GABA; hippocampus
5.  Sucrose Ingestion Induces Rapid AMPA Receptor Trafficking 
The mechanisms by which natural rewards such as sugar affect synaptic transmission and behavior are largely unexplored. Here, we investigate regulation of nucleus accumbens synapses by sucrose intake. Previous studies have shown that AMPA receptor trafficking is a major mechanism for regulating synaptic strength, and that in vitro, trafficking of AMPA receptors containing the GluA1 subunit takes place by a two-step mechanism involving extrasynaptic and then synaptic receptor transport. We report that in rat, repeated daily ingestion of a 25% sucrose solution transiently elevated spontaneous locomotion and potentiated accumbens core synapses through incorporation of Ca2+-permeable AMPA receptors (CPARs), which are GluA1-containing, GluA2-lacking AMPA receptors. Electrophysiological, biochemical and quantitative electron microscopy studies revealed that sucrose training (7 days) induced a stable (>24 hr) intraspinous GluA1 population, and that in these rats a single sucrose stimulus rapidly (5 min) but transiently (<24 hr) elevated GluA1 at extrasynaptic sites. CPARs and dopamine D1 receptors were required in vivo for elevated locomotion after sucrose ingestion. Significantly, a 7-day protocol of daily ingestion of a 3% solution of saccharin, a non-caloric sweetener, induced synaptic GluA1 similarly to 25% sucrose ingestion. These findings identify multi-step GluA1 trafficking, previously described in vitro, as a mechanism for acute regulation of synaptic transmission in vivo by a natural orosensory reward. Trafficking is stimulated by a chemosensory pathway that is not dependent on the caloric value of sucrose.
PMCID: PMC3767387  PMID: 23554493
6.  Adolescent female rats exhibiting activity-based anorexia express elevated levels of GABAA receptor α4 and δ subunits at the plasma membrane of hippocampal CA1 spines 
Synapse (New York, N.y.)  2012;66(5):391-407.
Activity-based anorexia (ABA) is an animal model for anorexia nervosa that has revealed genetic links to anxiety traits and neurochemical characteristics within the hypothalamus. However, few studies have used this animal model to investigate the biological basis for vulnerability of pubertal and adolescent females to ABA, even though the great majority of the anorexia nervosa cases are females exhibiting the first symptoms during puberty. GABAergic inhibition of the hippocampus strongly regulates anxiety as well as plasticity throughout life. We recently showed that the hippocampal CA1 of female mice undergo a dramatic change at puberty onset – from expressing virtually none of the non-synaptic α4βδ GABAA receptors (GABARs) pre-pubertally to expressing these GABARs at approximately 7% of the CA1 dendritic spine membranes at puberty onset. Furthermore, we showed that this change underlies the enhanced modulation of anxiety, neuronal excitability and NMDA receptor-dependent synaptic plasticity in the hippocampus by the stress neurosteroid, THP (3a-OH-5α[β]-pregnan-20-one or [allo]pregnanolone). Here, we used quantitative electron microscopy to determine whether ABA induction in female rats during adolescence also elevates the expression of α4 and δ subunits of α4βδ GABARs, as was observed at puberty onset for mice. Our analysis revealed that rats also exhibit a rise of α4 and δ subunits of α4βδ GABARs at puberty onset, in that these subunits are detectable at approximately 6% of the dendritic spine membranes of CA1 pyramidal cells at puberty onset (postnatal day 32–36; P32–36) but this drops to about 2% by P40 – P44. The levels of α4 and δ subunits at the CA1 spines remained low following exposure of P40 females to either of the two environmental factors needed to generate ABA - food restriction and access to a running wheel for four days (P40 to P44). This pattern contrasted greatly from those of ABA animals, for which the two environmental factors were combined. Within the hippocampus of ABA animals, 12% of the spine profiles were labeled for α4, reflecting a six-fold increase, relative to hippocampi of age-matched (P44) control females [p<0.005]. Concurrently, 7% of the spine profiles were labeled for δ, reflecting a 130% increase from the control values of 3% (p=0.01). No measurable change was detected for spine size. The observed magnitude of increase in the α4 and δ subunits at spines is sufficient to increase both tonic inhibition of hippocampus and anxiety during stress, thereby likely to exacerbate hyperactivity and weight loss.
PMCID: PMC3292704  PMID: 22213233
anorexia nervosa; receptor trafficking; anxiety; mood; stress; food restriction; hippocampus; hyperactivity; exercise; tonic inhibition; puberty; adolescence; eating disorder; allopregnanolone; pregnanolone; progesterone; THP; hippocampus; electron microscopic immunocytochemistry
7.  Knockout of the γ-aminobutyric acid receptor subunit α4 reduces functional δ-containing extrasynaptic receptors in hippocampal pyramidal cells at the onset of puberty 
Brain Research  2012;1450:11-23.
Increased plasmalemmal localization of α4βδ GABAA receptors (GABARs) occurs in the hippocampal pyramidal cells of female mice at pubertal onset (Shen et al., 2010). This increase occurs on both dendritic spines and shafts of CA1 pyramidal cells and is in response to hormone fluctuations that occur at pubertal onset. However, little is known about how the α4 and δ subunits individually mediate the formation of functional, plasmalemmal α4βδ GABARs. To determine whether expression of the α4 subunit is necessary for plasmalemmal δ subunit localization at pubertal onset, electron microscopic-immunocytochemistry (EM-ICC) was employed. CA1 pyramidal cells of female α4 knockout (KO) mice were tested for plasmalemmal levels of the δ subunit within dendritic spine and shaft profiles at the onset of puberty. EM-ICC revealed that the α4 and δ subunits localize on dendritic spines and shafts at sites extrasynaptic to GABAergic input at pubertal onset in tissue of wild-type (WT) mice. At pubertal onset, plasmalemmal localization of the δ subunit is reduced 45.9% on dendritic spines, and 56.7% on dendritic shafts with KO of the α4 subunit, as compared to WT tissue, yet levels of intracellular δ immunoreactivity remain unchanged. The decline in plasmalemmal localization is manifested as decreased responsiveness to the GABA agonist gaboxadol at concentrations that are selective for δ-containing GABARs. Additionally, α4 KO mice have larger dendritic spine and shaft profiles. Our findings demonstrate that α4 subunit expression strongly influences the pubertal increase of δ subunits at the plasma membrane, and that genetic deletion of α4 serves as a functional knock-down of δ-containing GABARs.
PMCID: PMC3319511  PMID: 22418059
Puberty; GABA type A receptor; THP; tonic inhibition; alpha 4; delta; allopregnanolone
8.  Endogenous GluR1-Containing AMPA Receptors Translocate to Asymmetric Synapses in the Lateral Amygdala During the Early Phase of Fear Memory Formation: An Electron Microscopic Immunocytochemical Study 
The Journal of comparative neurology  2010;518(23):4723-4739.
Although glutamate receptor 1 (GluR1)-containing α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate receptors (GluR1-AMPARs) are implicated in synaptic plasticity, it has yet to be demonstrated whether endogenous GluR1-AMPARs undergo activity-dependent trafficking in vivo to synapses to support short-term memory (STM) formation. The paradigm of pavlovian fear conditioning (FC) can be used to address this question, because a discrete region—the lateral amygdala (LA)—has been shown unambiguously to be necessary for the formation of the associative memory between a neutral stimulus (tone [CS]) and a noxious stimulus (foot shock [US]). Acquisition of STM for FC can occur even in the presence of protein synthesis inhibitors, indicating that redistribution of pre-existing molecules to synaptic junctions underlies STM. We employed electron microscopic immunocytochemistry to evaluate alterations in the distribution of endogenous AMPAR subunits at LA synapses during the STM phase of FC. Rats were sacrificed 40 minutes following three CS-US pairings. In the LA of paired animals, relative to naïve animals, the proportion of GluR1-AMPAR-labeled synapses increased 99% at spines and 167% in shafts. In the LA of unpaired rats, for which the CS was never associated with the US, GluR1 immunoreactivity decreased 84% at excitatory shaft synapses. GluR2/3 immunoreactivity at excitatory synapses did not change detectably following paired or unpaired conditioning. Thus, the early phase of FC involves rapid redistribution specifically of the GluR1-AMPARs to the postsynaptic membranes in the LA, together with the rapid translocation of GluR1-AMPARs from remote sites into the spine head cytoplasm, yielding behavior changes that are specific to stimulus contingencies.
PMCID: PMC3613289  PMID: 20963825
receptor trafficking; synaptic plasticity; pavlovian; fear conditioning; auditory cue; conditioned inhibition; endogenous gene products; associative memory; STM; LTM; stimulus contingencies
9.  The Biochemical Anatomy of Cortical Inhibitory Synapses 
PLoS ONE  2012;7(6):e39572.
Classical electron microscopic studies of the mammalian brain revealed two major classes of synapses, distinguished by the presence of a large postsynaptic density (PSD) exclusively at type 1, excitatory synapses. Biochemical studies of the PSD have established the paradigm of the synapse as a complex signal-processing machine that controls synaptic plasticity. We report here the results of a proteomic analysis of type 2, inhibitory synaptic complexes isolated by affinity purification from the cerebral cortex. We show that these synaptic complexes contain a variety of neurotransmitter receptors, neural cell-scaffolding and adhesion molecules, but that they are entirely lacking in cell signaling proteins. This fundamental distinction between the functions of type 1 and type 2 synapses in the nervous system has far reaching implications for models of synaptic plasticity, rapid adaptations in neural circuits, and homeostatic mechanisms controlling the balance of excitation and inhibition in the mature brain.
PMCID: PMC3387162  PMID: 22768092
10.  Puberty, steroids and GABAA receptor plasticity 
Psychoneuroendocrinology  2009;34S1:S91-S103.
GABAA receptors (GABAR) mediate most inhibition in the CNS and are also a target for neuroactive steroids such as 3α,5[α]β-THP (3αOH-5[α]β-OH-pregnan-20-one or [allo]pregnanolone). Although these steroids robustly enhance current gated by α1β2δ GABAR, we have shown that 3α,5[α]β-THP effects at recombinant α4β2δ GABAR depend on the direction of Cl- flux, where the steroid increases outward flux, but decreases inward flux through the receptor. This polarity-dependent inhibition of α4β2δ GABAR resulted from an increase in the rate and extent of rapid desensitization of the receptor, recorded from recombinant receptors expressed in HEK-293 cells with whole cell voltage clamp techniques. This inhibitory effect of 3α,5[α]β-THP was not observed at other receptor subtypes, suggesting it was selective for α4β2δ GABAR. Furthermore, it was prevented by a selective mutation of basic residue arginine 353 in the intracellular loop of the receptor, suggesting that this might be a putative chloride modulatory site. Expression of α4βδ GABAR increases markedly at extrasynaptic sites at the onset of puberty in female mice. At this time, 3α,5[α]β–THP decreased the inhibitory tonic current, recorded with perforated patch techniques to maintain the physiological Cl- gradient. By decreasing this shunting inhibition, 3α,5[α]β–THP increased the excitability of CA1 hippocampal pyramidal cells at puberty: These effects of the steroid were opposite to those observed before puberty when 3α,5[α]β–THP reduced neuronal excitability as a pre-synaptic effect. Behaviorally, the excitatory effect of 3α,5[α]β–THP was reflected as an increase in anxiety at the onset of puberty in female mice. Taken together, these findings suggest that the emergence of α4β2δ GABAR at the onset of puberty reverses the effect of a stress steroid. These findings may be relevant for the mood swings and increased response to stressful events reported in adolescence.
PMCID: PMC2794901  PMID: 19523771
pregnanolone; allopregnanolone; puberty; GABA-A receptor; neurosteroid; stress; chloride; anxiety; tonic current; CA1 hippocampus; steroid withdrawal
11.  Drebrin A Knockout Eliminates the Rapid Form of Homeostatic Synaptic Plasticity at Excitatory Synapses of Intact Adult Cerebral Cortex 
Homeostatic synaptic plasticity (HSP) is important for maintaining neurons' excitability within the dynamic range and for protecting neurons from unconstrained LTP that can cause breakdown of synapse specificity (Turrigiano, 2008). Knowledge of the molecular mechanism underlying this phenomenon remains incomplete, especially for the rapid form of HSP. In order to test whether HSP in adulthood depends on an F-actin binding protein – drebrin A, mice deleted of the adult isoform of drebrin (DAKO) but retaining the embryonic isoform (drebrin E) were generated. HSP was assayed by determining whether the NR2A subunit of NMDA receptors (NMDARs) can rise rapidly within spines following the application of an NMDAR antagonist, D-APV, onto the cortical surface. Electron microscopic immunocytochemistry revealed that, as expected, the D-APV treatment of WT mouse cortex increased the proportion of NR2A-immunolabeled spines within 30 min, relative to basal levels in hemispheres treated with an inactive enantiomer, L-APV. This difference was significant at the postsynaptic membrane and postsynaptic density (i.e., synaptic junction) as well as at non-synaptic sites within spines and was not accompanied by spine size changes. In contrast, the D-APV treatment of DAKO brains did not augment NR2A labeling within the spine cytoplasm or at the synaptic junction, even though basal levels of NR2A were not significantly different from those of WT cortices. These findings indicate that drebrin A is required for the rapid (<30 min) form of HSP at excitatory synapses of adult cortices while drebrin E is sufficient for maintaining basal NR2A levels within spines.
PMCID: PMC2839874  PMID: 19711416
12.  A Critical Role for α4βδ GABAA Receptors in Shaping Learning Deficits at Puberty in Mice 
Science (New York, N.Y.)  2010;327(5972):1515-1518.
The onset of puberty defines a developmental stage when some learning processes are diminished, but the mechanism for this deficit remains unknown. We found that, at puberty, expression of inhibitory α4βδ γ-aminobutyric acid type A (GABAA) receptors (GABAR) increases perisynaptic to excitatory synapses in CA1 hippocampus. Shunting inhibition via these receptors reduced N-methyl-D-aspartate receptor activation, impairing induction of long-term potentiation (LTP). Pubertal mice also failed to learn a hippocampal, LTP-dependent spatial task that was easily acquired by δ−/− mice. However, the stress steroid THP (3αOH-5α[β]-pregnan-20-one), which reduces tonic inhibition at puberty, facilitated learning. Thus, the emergence of α4βδ GABARs at puberty impairs learning, an effect that can be reversed by a stress steroid.
PMCID: PMC2887350  PMID: 20299596
13.  Gain Modulation by Nicotine in Macaque V1 
Neuron  2007;56(4):701-713.
Acetylcholine is a ubiquitous cortical neuromodulator implicated in cognition. In order to understand the potential for acetylcholine to play a role in visual attention, we studied nicotinic acetylcholine receptor (nAChR) localization and function in area V1 of the macaque. We found nAChRs presynaptically at thalamic synapses onto excitatory, but not inhibitory, neurons in the primary thalamorecipient layer 4c. Furthermore, consistent with the release enhancement suggested by this localization, we discovered that nicotine increases responsiveness and lowers contrast threshold in layer 4c neurons. We also found that nAChRs are expressed by GABAergic interneurons in V1 but rarely by pyramidal neurons, and that nicotine suppresses visual responses outside layer 4c. All sensory systems incorporate gain control mechanisms, or processes which dynamically alter input/output relationships. We demonstrate that at the site of thalamic input to visual cortex, the effect of this nAChR-mediated gain is an enhancement of the detection of visual stimuli.
PMCID: PMC2875676  PMID: 18031686
14.  Characterization of Age-Dependent and Progressive Cortical Neuronal Degeneration in Presenilin Conditional Mutant Mice 
PLoS ONE  2010;5(4):e10195.
Presenilins are the major causative genes of familial Alzheimer's disease (AD). Our previous study has demonstrated essential roles of presenilins in memory and neuronal survival. Here, we explore further how loss of presenilins results in age-related, progressive neurodegeneration in the adult cerebral cortex, where the pathogenesis of AD occurs. To circumvent the requirement of presenilins for embryonic development, we used presenilin conditional double knockout (Psen cDKO) mice, in which presenilin inactivation is restricted temporally and spatially to excitatory neurons of the postnatal forebrain beginning at 4 weeks of age. Increases in the number of degenerating (Fluoro-Jade B+, 7.6-fold) and apoptotic (TUNEL+, 7.4-fold) neurons, which represent ∼0.1% of all cortical neurons, were first detected at 2 months of age when there is still no significant loss of cortical neurons and volume in Psen cDKO mice. By 4 months of age, significant loss of cortical neurons (∼9%) and gliosis was found in Psen cDKO mice. The apoptotic cell death is associated with caspase activation, as shown by increased numbers of cells immunoreactive for active caspases 9 and 3 in the Psen cDKO cortex. The vulnerability of cortical neurons to loss of presenilins is region-specific with cortical neurons in the lateral cortex most susceptible. Compared to the neocortex, the increase in apoptotic cell death and the extent of neurodegeneration are less dramatic in the Psen cDKO hippocampus, possibly in part due to increased neurogenesis in the aging dentate gyrus. Neurodegeneration is also accompanied with mitochondrial defects, as indicated by reduced mitochondrial density and altered mitochondrial size distribution in aging Psen cortical neurons. Together, our findings show that loss of presenilins in cortical neurons causes apoptotic cell death occurring in a very small percentage of neurons, which accumulates over time and leads to substantial loss of cortical neurons in the aging brain. The low occurrence and significant delay of apoptosis among cortical neurons lacking presenilins suggest that loss of presenilins may induce apoptotic neuronal death through disruption of cellular homeostasis rather than direct activation of apoptosis pathways.
PMCID: PMC2855368  PMID: 20419112
15.  The Glutamate Transporter GLT1a Is Expressed in Excitatory Axon Terminals of Mature Hippocampal Neurons 
GLT1 is the major glutamate transporter of the brain and has been thought to be expressed exclusively in astrocytes. Although excitatory axon terminals take up glutamate, the transporter responsible has not been identified. GLT1 is expressed in at least two forms varying in the C termini, GLT1a and GLT1b. GLT1 mRNA has been demonstrated in neurons, without associated protein. Recently, evidence has been presented, using specific C terminus-directed antibodies, that GLT1b protein is expressed in neurons in vivo. These data suggested that the GLT1 mRNA detected in neurons encodes GLT1b and also that GLT1b might be the elusive presynaptic transporter. To test these hypotheses, we used variant-specific probes directed to the 3′-untranslated regions for GLT1a and GLT1b to perform in situ hybridization in the hippocampus. Contrary to expectation, GLT1a mRNA was the more abundant form. To investigate further the expression of GLT1 in neurons in the hippocampus, antibodies raised against the C terminus of GLT1a and against the N terminus of GLT1, found to be specific by testing in GLT1 knock-out mice, were used for light microscopic and EM-ICC. GLT1a protein was detected in neurons, in 14–29% of axons in the hippocampus, depending on the region. Many of the labeled axons formed axo-spinous, asymmetric, and, thus, excitatory synapses. Labeling also occurred in some spines and dendrites. The antibody against the N terminus of GLT1 also produced labeling of neuronal processes. Thus, the originally cloned form of GLT1, GLT1a, is expressed as protein in neurons in the mature hippocampus and may contribute significantly to glutamate uptake into excitatory terminals.
PMCID: PMC2849838  PMID: 14762132
uptake; trafficking; alternative splicing; excitotoxicity; PDZ domain; synapse
16.  Expression of a Variant Form of the Glutamate Transporter GLT1 in Neuronal Cultures and in Neurons and Astrocytes in the Rat Brain 
To identify glutamate transporters expressed in forebrain neurons, we prepared a cDNA library from rat forebrain neuronal cultures, previously shown to transport glutamate with high affinity and capacity. Using this library, we cloned two forms, varying in the C terminus, of the glutamate transporter GLT1. This transporter was previously found to be localized exclusively in astrocytes in the normal mature brain. Specific antibodies against the C-terminal peptides were used to show that forebrain neurons in culture express both GLT1a and GLT1b proteins. The pharmacological properties of glutamate transport mediated by GLT1a and GLT1b expressed in COS-7 cells and in neuronal cultures were indistinguishable. Both GLT1a and GLT1b were upregulated in astrocyte cultures by exposure to dibutyryl cAMP. We next investigated the expression of GLT1b in vivo. Northern blot analysis of forebrain RNA revealed two transcripts of ~3 and 11 kb that became more plentiful with developmental age. Immunoblot analysis showed high levels of expression in the cortex, hippocampus, striatum, thalamus, and midbrain. Pre-embedding electron microscopic immunocytochemistry with silver-enhanced immunogold detection was used to localize GLT1b in vivo. In the rat somatosensory cortex, GLT1b was clearly expressed in neurons in presynaptic terminals and dendritic shafts, as well as in astrocytes. The presence of GLT1b in neurons may offer a partial explanation for the observed uptake of glutamate by presynaptic terminals, for the preservation of input specificity at excitatory synapses, and may play a role in the pathophysiology of excitotoxicity.
PMCID: PMC2849837  PMID: 11896154
glutamate; transport; dihydrokainate; presynaptic; astrocytes; synapse; excitotoxicity
17.  Muscarinic Acetylcholine Receptors in Macaque V1 Are Most Frequently Expressed by Parvalbumin-Immunoreactive Neurons 
The Journal of comparative neurology  2008;507(5):1748-1762.
Acetylcholine (ACh) is believed to underlie mechanisms of arousal and attention in mammals. ACh also has a demonstrated functional effect in visual cortex that is both diverse and profound. We have reported previously that cholinergic modulation in V1 of the macaque monkey is strongly targeted toward GABAergic interneurons. Here we examine the localization of m1 and m2 muscarinic receptor subtypes across subpopulations of GABAergic interneurons—identified by their expression of the calcium-binding proteins parvalbumin, calbindin, and calretinin—using dual-immunofluorescence confocal microscopy in V1 of the macaque monkey. In doing so, we find that the vast majority (87%) of parvalbumin-immunoreactive neurons express m1-type muscarinic ACh receptors. m1 receptors are also expressed by 60% of calbindin-immunoreactive neurons and 40% of calretinin-immunoreactive neurons. m2 AChRs, on the other hand, are expressed by only 31% of parvalbumin neurons, 23% of calbindin neurons, and 25% of calretinin neurons. Parvalbumin-immunoreactive cells comprise ≈75% of the inhibitory neuronal population in V1 and included in this large subpopulation are neurons known to veto and regulate the synchrony of principal cell spiking. Through the expression of m1 ACh receptors on nearly all of these PV cells, the cholinergic system avails itself of powerful control of information flow through and processing within the network of principal cells in the cortical circuit.
PMCID: PMC2845159  PMID: 18265004
cholinergic; neuromodulation; GABAergic; striate cortex; immunofluorescence; dual-labeling; calcium-binding proteins; calbindin; calretinin; parvalbumin
18.  Optimization of differential immunogold-silver and peroxidase labeling with maintenance of ultrastructure in brain sections before plastic embedding 
Journal of neuroscience methods  1990;33(2-3):113-127.
The limited success of immunogold labeling for pre-embedding immunocytochemistry of neuronal antigens is largely attributed to poor penetration of large (5–20 nm) colloidal gold particles. We examined the applicability of using silver intensification of 1 nm colloidal gold particles non-covalently bound to goat anti-rabbit immunoglobulin (1) for single labeling of a rabbit antiserum against the catecholamine synthesizing enzyme, tyrosine hydroxylase (TH), and (2) for immunogold localization of rabbit anti-TH simultaneously with immunoperoxidase labeling of a mouse monoclonal antibody against the opiate peptide, leucine-enkephalin (LE). Vibratome sections were collected from acrolein fixed brains of adult rats. These sections were immunolabeled without use of freeze-thawing or other methods that enhance penetration, but damage ultrastructure. By light microscopy, incubations in the silver intensifier (Intense M, Janssen) for less than 10 min at room temperature resulted in a brownish-red reaction product for TH. This product was virtually indistinguishable from that seen using diaminobenzidine reaction for detection of peroxidase immunoreactivity. Longer incubations produced intense black silver deposits that were more clearly distinguishable from the brown immunoperoxidase labeling. However, by light microscopy, the gold particles seen by electron microscopy were most readily distinguished from peroxidase reaction product with shorter silver intensification periods. The smaller size of gold particles with shorter periods of silver intensification also facilitated evaluation of labeling with respect to subcellular organdies. Detection of the silver product did not appear to be appreciably changed by duration of post-fixation in osmium tetroxide. In dual-labeled sections, perikarya and terminals exhibiting immunogold-silver labeling for TH were distinct from those containing immunoperoxidase labeling for LE. These results (1) define the conditions needed for optimal immunogold-silver labeling of antigens while maintaining the ultrastructural morphology in brain, and (2) establish the necessity for controlled silver intensification for light or electron microscopic differentiation of immunogold-silver and peroxidase reaction products and for optimal subcellular resolution.
PMCID: PMC2845158  PMID: 1977960
Tyrosine hydroxylase; Enkephalin; Double labeling; Silver enhancement; Catecholamine
19.  Drebrin A Content Correlates With Spine Head Size in the Adult Mouse Cerebral Cortex 
Synaptic activities alter synaptic strengths at the axospinous junctions, and such changes are often accompanied by changes in the size of the postsynaptic spines. We have been exploring the idea that drebrin A, a neuron-specific actin-binding protein localized on the postsynaptic side of excitatory synapses, may be a molecule that links synaptic activity to the shape and content of spines. Here, we performed electron microscopic immunocytochemistry with the nondiffusible gold label to explore the relationship among levels of drebrin A, the NR2A subunit of N-methyl-D-aspartate receptors, and the size of spines in the perirhinal cortex of adult mouse brains. In contrast to the membranous localization within neonatal spines, most immunogold particles for drebrin A were localized to the cytoplasmic core region of spines in mature spines. This distribution suggests that drebrin within adult spines may reorganize the F-actin network at the spine core, in addition to its known neonatal role in spine formation. Drebrin A-immunopositive (DIP) spines exhibited larger spine head areas and longer postsynaptic densities (PSDs) than drebrin A-immunonegative (DIN) spines (P < 0.001). Furthermore, spine head area and PSD lengths correlated positively with drebrin A levels (r = 0.47 and 0.40). The number of synaptic NR2A immunolabels was also higher in DIP spines than in DIN spines, whereas their densities per unit lengths of PSD were not significantly different. These differences between the DIP and the DIN spines indicate that spine sizes and synaptic protein composition of mature brains are regulated, at least in part, by drebrin A levels.
PMCID: PMC2844454  PMID: 17559090
electron microscopy; dendritic spine; immunocytochemistry; actin binding protein; postsynaptic density; NMDA receptor
20.  Chemical and Morphological Alterations of Spines Within the Hippocampus and Entorhinal Cortex Precede the Onset of Alzheimer’s Disease Pathology in Double Knock-In Mice 
Mice with knock-in of two mutations that affect beta amyloid processing and levels (2xKI) exhibit impaired spatial memory by 9–12 months of age, together with synaptic plasticity dysfunction in the hippocampus. The goal of this study was to identify changes in the molecular and structural characteristics of synapses that precede and thus could exert constraints upon cellular mechanisms underlying synaptic plasticity. Drebrin A is one protein reported to modulate spine sizes and trafficking of proteins to and from excitatory synapses. Thus, we examined levels of drebrin A within postsynaptic spines in the hippocampus and entorhinal cortex. Our electron microscopic immunocytochemical analyses reveal that, by 6 months, the proportion of hippocampal spines containing drebrin A is reduced and this change is accompanied by an increase in the mean size of spines and decreased density of spines. In the entorhinal cortex of 2xKI brains, we detected no decrement in the proportion of spines labeled for drebrin A and no significant change in spine density at 6 months, but rather a highly significant reduction in the level of drebrin A immunoreactivity within each spine. These changes are unlike those observed for the somatosensory cortex of 2xKI mice, in which synapse density and drebrin A immunoreactivity levels remain unchanged at 6 months and older. These results indicate that brains of 2xKI mice, like those of humans, exhibit regional differences of vulnerability, with the hippocampus exhibiting the first signatures of structural changes that, in turn, may underlie the emergent inability to update spatial memory in later months.
PMCID: PMC2844449  PMID: 17912741
amyloid precursor protein; presenilin 1; drebrin; excitatory synapses; electron microscopic immunocytochemistry; ultrastructure
21.  Plasticity in Brain Development 
Scientific American  1988;259(6):56-64.
The final wiring of the brain occurs after birth and is governed by early experience. A protein called MAP2 seems to take part in the molecular events that underlie the brain’s ability to change
PMCID: PMC2841150  PMID: 2849807
22.  α7 Nicotinic Acetylcholine Receptors Occur at Postsynaptic Densities of AMPA Receptor-Positive and -Negative Excitatory Synapses in Rat Sensory Cortex 
NMDA receptor (NMDAR) activation requires concurrent membrane depolarization, and glutamatergic synapses lacking AMPA receptors (AMPARs) are often considered “silent” in the absence of another source of membrane depolarization. During the second postnatal week, NMDA currents can be enhanced in rat auditory cortex through activation of the α7 nicotinic acetylcholine receptor (α7nAChR). Electrophysiological results support a mainly presynaptic role for α7nAChR at these synapses. However, immunocytochemical evidence that α7nAChR is prevalent at postsynaptic sites of glutamatergic synapses in hippocampus and neocortex, along with emerging electrophysiological evidence for postsynaptic nicotinic currents in neocortex and hippocampus, has prompted speculation that α7nAChR allows for activation of NMDAR postsynaptically at synapses lacking AMPAR. Here we used dual immunolabeling and electron microscopy to examine the distribution of α7nAChR relative to AMPAR (GluR1, GluR2, and GluR3 subunits combined) at excitatory synapses in somatosensory cortex of adult and 1-week-old rats. α7nAChR occurred discretely over most of the thick postsynaptic densities in all cortical layers of both age groups. AMPAR immunoreactivity was also detectable at most synapses; its distribution was independent of that of α7nAChR. In both age groups, approximately one-quarter of asymmetrical synapses were α7nAChR positive and AMPAR negative. The variability of postsynaptic α7nAChR labeling density was greater at postnatal day (PD) 7 than in adulthood, and PD 7 neuropil contained a subset of small AMPA receptor-negative synapses with a high density of α7nAChR immunoreactivity. These observations support the idea that acetylcholine receptors can aid in activating glutamatergic synapses and work together with AMPA receptors to mediate postsynaptic excitation throughout life.
PMCID: PMC2839916  PMID: 12077196
sensory cortex; receptive field properties; α7 nicotinic acetylcholine receptor; glutamate; AMPA receptor; NMDA receptor; synaptic plasticity; early postnatal development; postsynaptic density; immunocytochemistry; electron microscopy
23.  Silencing of Neuroligin Function by Postsynaptic Neurexins 
The formation of neuronal circuits during development involves a combination of synapse stabilization and elimination events. Synaptic adhesion molecules are thought to play an important role in synaptogenesis, and several trans-synaptic adhesion systems that promote the formation and maturation of synapses have been identified. The neuroligin–neurexin complex is a heterophilic adhesion system that promotes assembly and maturation of synapses through bidirectional signaling. In this protein complex, postsynaptic neuroligins are thought to interact trans-synaptically with presynaptic neurexins. However, the subcellular localization of neurexins has not been determined. Using immunoelectron microscopy, we found that endogenous neurexins and epitope-tagged neurexin-1β are localized to axons and presynaptic terminals in vivo. Unexpectedly, neurexins are also abundant in the postsynaptic density. cis-expression of neurexin-1β with neuroligin-1 inhibits trans-binding to recombinant neurexins, blocks the synaptogenic activity of neuroligin-1, and reduces the density of presynaptic terminals in cultured hippocampal neurons. Our results demonstrate that the function of neurexin proteins is more diverse than previously anticipated and suggest that postsynaptic cis-interactions might provide a novel mechanism for silencing the activity of a synaptic adhesion complex.
PMCID: PMC2839889  PMID: 17360903
synapse formation; hippocampus; adhesion; neuroligin; neurexin; hippocampal neurons
24.  Columnar activity regulates astrocytic β-adrenergic receptor-like immunoreactivity in V1 of adult monkeys 
Visual neuroscience  1994;11(1):179-187.
Recent results indicate that astrocytic β-adrenergic receptors (βAR) participate in noradrenergic modulation of synaptic activity. In this study, we sought to examine whether neural activity can, in turn, regulate astrocytic βAR. To address this question, an antiserum that recognizes β-adrenergic receptors (βAR) specifically in astrocytes was used to assess the distribution of the receptors across ocular dominance columns in V1 of two monocular and four visually intact adult monkeys. Cytochrome oxidase histochemistry (CO) was used to identify the position of the cortical laminae and of the ocular dominance columns receiving visual inputs from the intact and enucleated eyes. This stain revealed the expected pattern within V1 of monocular monkeys –i.e. darker and lighter bands of equal widths (ca. 500 μm) spanning laminae 4–6, each associated with larger and smaller blobs, respectively, in lamina 2/3. Alignment of CO sections with adjacent sections stained for astrocytic βAR by the immunoperoxidase method revealed intense βAR-like immunoreactivity (βAR-li) in the superficial laminae, a slightly weaker staining in the infragranular laminae and weakest staining in lamina 4C. Within lamina 4C, a prominent striped pattern was evident. The darker bands of the stripe closely matched widths and positions of the lighter CO columns associated with the enucleated eye. On the other hand, immunocytochemical staining for the astrocytic intermediate filament protein, GFAP, within V1 of monocular monkeys revealed no inter-columnar difference in the density of astrocytic cell bodies or processes. Nissl stain also revealed no overt inter-columnar differences in cell density. V1 of visually intact monkeys exhibited a similar laminar distribution pattern of βAR-li and of CO. Within lamina 4C, βAR-li was uniformly faint and CO staining was uniformly intense. This suggests that the striped pattern of βAR-li seen in lamina 4C of monocular monkeys results from elevation of the βAR-antigen within the inactive columns. The results indicate that astrocytic βAR density is regulated by local neural activity. The mechanisms regulating βAR density are likely to be independent of those regulating glial cell proliferation or GFAP synthesis. In vitro experimental results of others suggest that elevation of astrocytic βAR may be a mechanism compensating for chronic neural inactivity, since the coincident release of noradrenaline with visual stimulation would elevate neuropil excitability via the astrocytic mechanism of (1) decreasing the uptake of neuronally released L-glutamate; (2) increasing GABA uptake; and (3) stimulating glycogenolysis. Alternatively, the changes in βAR-li may reflect an up-regulation of the receptors within inactive columns due to reduced levels of noradrenaline release.
PMCID: PMC2839236  PMID: 8011579
Astrocytes; Catecholamine receptors; Activity-dependent regulation; Monocular deprivation; V1
25.  β-Adrenergic Receptors: Astrocytic Localization in the Adult Visual Cortex and Their Relation to Catecholamine Axon Terminals as Revealed by Electron Microscopic Immunocytochemistry 
It has long been recognized that noradrenaline, the most abundant catecholamine within the visual cortex, plays important roles in modulating the sensitivity of cortical neurons to visual stimuli. However, whether or not these noradrenaline effects are confined to a discrete synaptic specialization or mediated by diffuse modulation of a group of synapses has remained an issue open for debate. The aim of this study was to examine the cellular basis for noradrenaline action within the visual cortex of adult rats and cats. To this end, I used electron microscopic immunocytochemistry to examine the relationship between (1) catecholamine axon terminals and β-adrenergic receptors (βAR), which, together, may define the effective sphere of noradrenaline modulation; and then (2) these putative sites for catecholamine modulation and axospinous asymmetric junctions where excitatory neurotransmission is likely to dominate. Antibodies against βAR were used at light and electron microscopic levels on the visual cortex of rat and cat. Rat visual cortex was also labeled simultaneously for βAR and the catecholamine-synthesizing enzyme, tyrosine hydroxylase (TH), to determine the ultrastructural relationships between catecholamine terminals and βAR. Immunoperoxidase labeling revealed that βAR404, a polyclonal antibody directed against the C-terminal tail of hamster lung βAR (β2-type), recognized astrocytic processes predominantly. In contrast, βAR248, a polyclonal antibody directed against the third cytoplasmic loop, recognized neuronal perikarya as observed in previous studies. Dual labeling for βAR404 and TH revealed that catecholamine axon terminals that contained numerous vesicles formed direct contacts with astrocytic processes exhibiting βAR404 immunoreactivity. However, some catecholamine axon terminals that lacked dense clusters of vesicles were positioned away from βAR404-immunoreactive astrocytes. Frequently, βAR-immunoreactive astrocytic processes surrounded asymmetric axospinous junctions while also contacting catecholamine axon terminals. These observations support the possibility that, through activation of astrocytic βAR, noradrenaline modulates astrocytic uptake mechanism for excitatory amino acids, such as L-glutamate. Astrocytic βAR might also define the effective sphere of catecholamine modulation through alterations in the morphology of distal astrocytic processes and the permeability of gap junctions formed between astrocytes.
PMCID: PMC2838201  PMID: 1347560

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