Baicalein reduces β‐amyloid and promotes nonamyloidogenic amyloid precursor protein processing in an Alzheimer's disease transgenic mouse model

Baicalein, a flavonoid isolated from the roots of Scutellaria baicalensis, is known to modulate γ‐aminobutyric acid (GABA) type A receptors. Given prior reports demonstrating benefits of GABA_A modulation for Alzheimer's disease (AD) treatment, we wished to determine whether this agent might be beneficial for AD. CHO cells engineered to overexpress wild‐type amyloid precursor protein (APP), primary culture neuronal cells from AD mice (Tg2576) and AD mice were treated with baicalein. In the cell cultures, baicalein significantly reduced the production of β‐amyloid (Aβ) by increasing APP α‐processing. These effects were blocked by the GABA_A antagonist bicuculline. Likewise, AD mice treated daily with i.p. baicalein for 8 weeks showed enhanced APP α‐secretase processing, reduced Aβ production, and reduced AD‐like pathology together with improved cognitive performance. Our findings suggest that baicalein promotes nonamyloidogenic processing of APP, thereby reducing Aβ production and improving cognitive performance, by activating GABA_A receptors. © 2013 Wiley Periodicals, Inc.     

Binding and uptake of Aβ1‐42 by primary human astrocytes in vitro

Clearance of the amyloid‐β peptide (Aβ) as a remedy for Alzheimer's disease (AD) is a major target in on‐going clinical trials. In vitro studies confirmed that Aβ is taken up by rodent astrocytes, but knowledge on human astrocyte‐mediated Aβ clearance is sparse. Therefore, by means of flow cytometry and confocal laser scanning microscopy (CLSM), we evaluated the binding and internalization of Aβ1‐42 by primary human fetal astrocytes and adult astrocytes, isolated from nondemented subjects (n = 8) and AD subjects (n = 6). Furthermore, we analyzed whether α1‐antichymotrypsin (ACT), which is found in amyloid plaques and can influence Aβ fibrillogenesis, affects the Aβ uptake by human astrocytes. Upon over night exposure of astrocytes to FAM‐labeled Aβ1‐42 (10 μM) preparations, (80.7 ± 17.7)% fetal and (52.9 ± 20.9)% adult Aβ‐positive astrocytes (P = 0.018) were observed. No significant difference was found in Aβ1‐42 uptake between AD and non‐AD astrocytes, and no influence of ApoE genotype on Aβ1‐42 uptake was observed in any group. There was no difference in the percentage of Aβ‐positive cells upon exposure to Aβ1‐42 (10 μM) combined with ACT (1,000:1, 100:1, and 10:1 molar ratio), versus Aβ1‐42 alone. CLSM revealed binding of Aβ1‐42 to the cellular surfaces and cellular internalization of smaller Aβ1‐42 fragments. Under these conditions, there was no increase in cellular release of the proinflammatory chemokine monocyte‐chemoattractant protein 1, as compared with nontreated control astrocytes. Thus, primary human astrocytes derived from different sources can bind and internalize Aβ1‐42, and fetal astrocytes were more efficient in Aβ1‐42 uptake than adult astrocytes. © 2008 Wiley‐Liss, Inc.     

Secreted amyloid precursor protein and holo‐APP bind amyloid β through distinct domains eliciting different toxic responses on hippocampal neurons

Amyloid β (Aβ) is a metabolic product of Aβ precursor protein (APP). Deposition of Aβ in the brain and neuronal degeneration are characteristic hallmarks of Alzheimer's disease (AD). Aβ induces neuronal degeneration, but the mechanism of neurotoxicity remains elusive. Increasing evidence implicates APP as a receptor‐like protein for Aβ fibrils (fAβ). In this study, we present further experimental support for the direct interaction of APP with fAβ and for its involvement in Aβ neurotoxicity. Using recombinant purified holo‐APP (h‐APP), we have shown that it directly binds fAβ. Employing deletion mutant forms of APP, we show that two different sequences are involved in the binding of APP to fAβ. One sequence in the n‐terminus of APP is required for binding of fAβ to secreted APP (s‐APP) but not to h‐APP. In addition, the extracellular juxtamembrane Aβ‐sequence mediates binding of fAβ to h‐APP but not to s‐APP. Deletion of the extracellular juxtamembrane Aβ sequence abolishes abnormal h‐APP accumulation and toxicity induced by fAβ deposition, whereas deletions in the n‐terminus of APP do not affect Aβ toxicity. These experiments show that interaction of toxic Aβ species with its membrane‐anchored parental protein promotes toxicity in hippocampal neurons, adding further support to an Aβ‐receptor‐like function of APP directly implicated in neuronal degeneration in AD. © 2010 Wiley‐Liss, Inc.     

Analysis of β‐amyloid (Aβ) deposition in the temporal lobe in Alzheimer's disease using Fourier (spectral) analysis

R. A. Armstrong and N. J. Cairns (2010) Neuropathology and Applied Neurobiology 36, 248–257
Analysis of β‐amyloid (Aβ) deposition in the temporal lobe in Alzheimer's disease using Fourier (spectral) analysis Aim: To determine the spatial pattern of β‐amyloid (Aβ) deposition throughout the temporal lobe in Alzheimer's disease (AD). Methods: Sections of the complete temporal lobe from six cases of sporadic AD were immunolabelled with antibody against Aβ. Fourier (spectral) analysis was used to identify sinusoidal patterns in the fluctuation of Aβ deposition in a direction parallel to the pia mater or alveus. Results: Significant sinusoidal fluctuations in density were evident in 81/99 (82%) analyses. In 64% of analyses, two frequency components were present with density peaks of Aβ deposits repeating every 500–1000 µm and at distances greater than 1000 µm. In 25% of analyses, three or more frequency components were present. The estimated period or wavelength (number of sample units to complete one full cycle) of the first and second frequency components did not vary significantly between gyri of the temporal lobe, but there was evidence that the fluctuations of the classic deposits had longer periods than the diffuse and primitive deposits. Conclusions: (i) Aβ deposits exhibit complex sinusoidal fluctuations in density in the temporal lobe in AD; (ii) fluctuations in Aβ deposition may reflect the formation of Aβ deposits in relation to the modular and vascular structure of the cortex; and (iii) Fourier analysis may be a useful statistical method for studying the patterns of Aβ deposition both in AD and in transgenic models of disease.     

Scavenging of Alzheimer's amyloid β-protein by microglia in culture

Deposits of amyloid β-protein (Aβ) form the cores of the pathological plaques which characterize Alzheimer's disease. The mechanism of formation of the deposits is unknown; one possibility is failure of a clearance mechanism that would normally remove the protein from brain parenchyma. This study has investigated the capacity of the central nervous system (CNS) phagocytes, microglia cells, to clear exogenous Aβ_1–42 from their environment. Cultured microglia from adult rat CNS have a high capacity to remove Aβ from serum-free medium, shown by immunoblotting experiments. Aβ from incubation medium was attached to the cell surface and could be identified by immunocytochemistry at the light or electron microscopic (EM) level; by EM, Aβ also appeared in phagosome-like intracellular vesicles. Light microscopic immunocytochemistry combined with computer-assisted image analysis showed that cells accumulated Aβ within 24 hr. from culture medium containing from 1 to 20 μg/ml Aβ. Microglial accumulation of Aβ was substantially reduced in the presence of fetal bovine serum. Addition of the protease inhibitor leupeptin to incubation medium with serum resulted in accumulation of Aβ in a membrane-bound intracellular compartment, but not at the cell surface. The increase in intracellular accumulation in the presence of the protease inhibitor indicates a microglial capacity for intracellular degradation of Aβ in the absence of inhibition. The change from predominantly cell-surface accumulation in serum-free medium to predominantly intracellular accumulation with serum may be explained by the presence in serum of carrier proteins that complex with Aβ and target it to cell surface receptors capable of stimulating endocytosis. Microglia were also cultured on unfixed cryostat sections of human brain tissue containing Alzheimer's plaques. Very little Aβ from the tissue was accumulated by the cells, although cultured microglia were found in direct contact with anti-Aβ immunopositive plaques. Possibly Aβ in tissue sections was complexed with other proteins which either inhibited its uptake by microglia or enhanced its proteolysis, preventing cellular accumulation of immunostainable Aβ. The results indicate that cultured microglia effectively remove Aβ from tissue culture medium and from the surface of the dish and concentrate monomer and aggregates of Aβ either on the cell surface or intracellularly. This process may be modified by proteins present in Alzheimer's brain sections. © 1996 Wiley-Liss, Inc.     

Enhanced aggregation and β structure of amyloid β peptide after coincubation with C1Q

Several lines of evidence now suggest that aggregation of soluble amyloid β peptide (Aβ) into a cross β sheet configuration may be an important factor in mediating potential neurotoxicity of Aβ. Synthetic Aβ has been shown to self aggregate in vitro. Here, we demonstrate that coincubation of freshly solubilized Aβ with C1q, a complement component known to bind Aβ in vitro and to colocalize with Aβ in vivo, results in as much as a 7-fold enhancement of Aβ aggregation, as well as a 2–4-fold enhancement of β structure within aggregates. The addition of C1q to preformed Aβ aggregates also results in significantly increased resistance to aggregate resolubilization. © 1994 Wiley-Liss, Inc.     

Cyclin‐Dependent kinase 5 targeting prevents β‐Amyloid aggregation involving glycogen synthase kinase 3β and phosphatases

Inappropriate activation of cyclin‐dependent kinase 5 (CDK5) resulting from proteolytic release of the activator fragment p25 from the membrane contributes to the formation of neurofibrillary tangles, β‐amyloid (βA) aggregation, and chronic neurodegeneration. At 18 months of age, 3× Tg‐AD mice were sacrificed after either 3 weeks (short term) or 1 year (long term) of CDK5 knockdown. In short‐term‐treated animals, CDK5 knockdown reversed βA aggregation in the hippocampi via inhibitory phosphorylation of glycogen synthase kinase 3β Ser9 and activation of phosphatase PP2A. In long‐term‐treated animals, CDK5 knockdown induced a persistent reduction in CDK5 and prevented βA aggregation, but the effect on amyloid precursor protein processing was reduced, suggesting that yearly booster therapy would be required. These findings further validate CDK5 as a target for preventing or blocking amyloidosis in older transgenic mice. © 2015 Wiley Periodicals, Inc.     

Limited expression of heparan sulphate proteoglycans associated with Aβ deposits in the APPswe/PS1dE9 mouse model for Alzheimer's disease

N. M. Timmer, M. K. Herbert, J. W. Kleinovink, A. J. Kiliaan, R. M. W. de Waal and M. M. Verbeek (2010) Neuropathology and Applied Neurobiology 36, 478–486
Limited expression of heparan sulphate proteoglycans associated with Aβ deposits in the APPswe/PS1dE9 mouse model for Alzheimer's disease Aims: Alzheimer's disease (AD) is characterized by deposition of the amyloid beta (Aβ) peptide in brain parenchyma and vasculature. Several proteins co‐deposit with Aβ, including heparan sulphate proteoglycans (HSPG). HSPG have been suggested to contribute to Aβ aggregation and deposition, and may influence plaque formation and persistence by stimulating Aβ fibrillization and by protecting Aβ against degradation. Mouse models for AD, expressing the human amyloid precursor protein (APP), produce Aβ deposits similar to humans. These models may be used to study disease pathology and to develop new therapeutic interventions. We aimed to investigate whether co‐deposition of HSPG in AD brains can be replicated in the APPswe/PS1dE9 mouse model for AD and if a temporal association of HSPG with Aβ exists. Methods: We studied the co‐deposition of several HSPG and of the glycosaminoglycan side chains of HSPG in the APPswe/PS1dE9 model at different ages by immunohistochemistry. Results: We found that, although APPswe/PS1dE9 mice did develop severe Aβ pathology with age, co‐deposition of HS glycosaminoglycan chains and the various HSPG (agrin, perlecan and glypican‐1) was scarce (<10–30% of the Aβ deposits were stained). Conclusions: Our data suggest that the molecular composition of Aβ deposits in the APPswe/PS1dE9 mouse, with respect to the several HSPG investigated in this study, does not accurately reflect the human situation. The near absence of HSPG in Aβ deposits in this transgenic mouse model may, in turn, hinder the translation of preclinical intervention studies from mice to men.     

Astrocytic Aβ1‐42 uptake is determined by Aβ‐aggregation state and the presence of amyloid‐associated proteins

Intracerebral accumulation of amyloid‐β (Aβ) leading to Aβ plaque formation, is the main hallmark of Alzheimer's disease and might be caused by defective Aβ‐clearance. We previously found primary human astrocytes and microglia able to bind and ingest Aβ1‐42 in vitro, which appeared to be limited by Aβ1‐42 fibril formation. We now confirm that astrocytic Aβ‐uptake depends on size and/or composition of Aβ‐aggregates as astrocytes preferably take up oligomeric Aβ over fibrillar Aβ. Upon exposure to either fluorescence‐labelled Aβ1‐42 oligomers (Aβ_oligo) or fibrils (Aβ_fib), a larger (3.7 times more) proportion of astrocytes ingested oligomers compared to fibrils, as determined by flow cytometry. Aβ‐internalization was verified using confocal microscopy and live‐cell imaging. Neither uptake of Aβ_oligo nor Aβ_fib, triggered proinflammatory activation of the astrocytes, as judged by quantification of interleukin‐6 and monocyte‐chemoattractant protein‐1 release. Amyloid‐associated proteins, including α1‐antichymotrypsin (ACT), serum amyloid P component (SAP), C1q and apolipoproteins E (ApoE) and J (ApoJ) were earlier found to influence Aβ‐aggregation. Here, astrocytic uptake of Aβ_fib increased when added to the cells in combination with SAP and C1q (SAP/C1q), but was unchanged in the presence of ApoE, ApoJ and ACT. Interestingly, ApoJ and ApoE dramatically reduced the number of Aβ_oligo‐positive astrocytes, whereas SAP/C1q slightly reduced Aβ_oligo uptake. Thus, amyloid‐associated proteins, especially ApoJ and ApoE, can alter Aβ‐uptake in vitro and hence may influence Aβ clearance and plaque formation in vivo. © 2010 Wiley‐Liss, Inc.     

The peripheral messenger RNA expression of glycogen synthase kinase‐3β genes in Alzheimer's disease patients: a preliminary study

Objective: To explore the peripheral leucocytic messenger RNA (mRNA) expression of glycogen synthase kinase‐3β (GSK‐3β) gene in Alzheimer's disease (AD) patients. Methods: Using TaqMan relative quantitative real‐time polymerase chain reaction, we analyzed leucocytic gene expression of GSK‐3β in 48 AD patients and 49 healthy controls. Clinical data of AD patients were also collected. Results: The mRNA expression level of the GSK‐3β gene was significantly higher in the AD group (3.13 ± 0.62) than in the normal group (2.77 ± 0.77). Correlational analyses showed that the mRNA expression level of GSK‐3β gene in AD patients was associated with the age of onset (P= 0.047), age (P= 0.055), and Behavioral Pathology in Alzheimer's Disease Rating Scale total score (P= 0.062) and subscores: aggressiveness score (P= 0.073) and anxieties and phobias score (P= 0.067). Through multivariate regression model, older age, higher anxieties and phobias score and aggressiveness score were associated with higher mRNA expression level of GSK‐3β gene. Conclusion: In AD patients, the mRNA expression level of the GSK‐3β gene is increased and may be related to age and behavioural pathology in AD.     

Dual pathways mediate β‐amyloid stimulated glutathione release from astrocytes

Oxidative stress plays an important role in the progression of Alzheimer's disease (AD) and other neurodegenerative conditions. Glutathione (GSH), the major antioxidant in the central nervous system, is primarily synthesized and released by astrocytes. We determined if β‐amyloid (Aβ42), crucially involved in Alzheimer's disease, affected GSH release. Monomeric Aβ (mAβ) stimulated GSH release from cultured cortical astrocytes more effectively than oligomeric Aβ (oAβ) or fibrillary Aβ (fAβ). Monomeric Aβ increased the expression of the transporter ABCC1 (also referred to as MRP1) that is the main pathway for GSH release. GSH release from astrocytes, with or without mAβ stimulation, was reduced by pharmacological inhibition of ABCC1. Astrocytes robustly express connexin proteins, especially connexin43 (Cx43), and mAβ also stimulated Cx43 hemichannel‐mediated glutamate and GSH release. Aβ‐stimulation facilitated hemichannel opening in the presence of normal extracellular calcium by reducing astrocyte cholesterol level. Aβ treatment did not alter the intracellular concentration of reduced or oxidized glutathione. Using a mouse model of AD with early onset Aβ deposition (5xFAD), we found that cortical ABCC1 was significantly increased in temporal register with the surge of Aβ levels in these mice. ABCC1 levels remained elevated from 1.5 to 3.5 months of age in 5xFAD mice, before plunging to subcontrol levels when amyloid plaques appeared. Similarly, in cultured astrocytes, prolonged incubation with aggregated Aβ, but not mAβ, reduced induction of ABCC1 expression. These results support the hypothesis that in the early stage of AD pathogenesis, less aggregated Aβ increases GSH release from astrocytes (via ABCC1 transporters and Cx43 hemichannels) providing temporary protection from oxidative stress which promotes AD development. GLIA 2015;63:2208–2219     

Active clearance of human amyloid β 1–42 peptide aggregates from the rat ventricular system

A major constituent of SP in the brains of Alzheimer's disease is 39–43 amino acid peptide called β‐amyloid peptide (Aβ). Recent data have demonstrated that Aβ has a strong tendency to form insoluble aggregates and that toxic effects of Aβ is based on its aggregation. In the current study, 100 µg of human synthetic Aβ 1–42 (sAβ 1–42) was infused into the lateral ventricle of rat brain using a short‐term infusion model. At 2 or 7 days following the infusion, sAβ 1–42 was found to form insoluble aggregates, scattering throughout the entire ventricular systems. The sAβ 1–42 aggregates were partially engulfed by phagocytic cells and deposited at the meningeal vessels or the choroid plexuses. However, these deposits mostly disappeared from the ventricles by 28 days post‐infusion. Here, it is reported for the first time that considerable amounts of sAβ 1–42 are almost cleared from the rat ventricular system by the mononuclear phagocytic system.     

Peripheral treatment with enoxaparin exacerbates amyloid plaque pathology in Tg2576 mice

Alzheimer's disease (AD) is a complex, progressive neurological disorder characterized by the formation of extracellular amyloid plaques composed of β‐amyloid protein (Aβ), the key component in pathogenesis of AD. Peripheral administration of enoxaparin (ENO) reportedly reduces the level of Aβ and the amyloid plaques in the cortex of amyloid precursor protein (APP) transgenic mice. However, the exact mechanism of these effects is unclear. Our previous studies indicated that ENO can inhibit APP processing to Aβ in primary cortical cells from Tg2576 mice by downregulating BACE1 levels. This study examines whether ENO‐induced reduction of amyloid load is due to the decreased APP processing to Aβ in Tg2576 mice. Surprisingly, our results indicated that ENO significantly increases the Aβ42/Aβ40 ratio in cortex and enhances the amyloid plaque load in both cortex and hippocampus, although overall APP processing was not influenced by ENO. Moreover, ENO stimulated the aggregation of both Aβ40 and Aβ42 in vitro. Although ENO has been reported to improve cognition in vivo and has potential as a therapeutic agent for AD, the results from our study suggest that ENO can exacerbate the amyloid pathology, and the strategy of using ENO for the treatment of AD may require further assessment. © 2016 Wiley Periodicals, Inc.     

β‐amyloid and postural instability and gait difficulty in Parkinson's disease at risk for dementia

Although motor impairments in Parkinson's disease (PD) are attributed to nigrostriatal dopaminergic denervation, postural instability and gait difficulty (PIGD) features are less responsive to dopaminergic medications. PIGD features are a risk factor also for the development of dementia in PD (PDD). These observations suggest that nondopaminergic mechanisms may contribute to axial motor impairments. The aim was to perform a correlative PET study to examine the relationship between neocortical β‐amyloid deposition ([¹¹C]‐Pittsburgh Compound B), nigrostriatal dopaminergic denervation ([¹¹C]‐dihydrotetrabenazine), and PIGD feature severity in PD patients at risk for dementia. This was a cross‐sectional study of 44 PD patients (11 female and 33 male; 69.5 ± 6.6 years of age; 7.0 ± 4.8 years motor disease duration; mean H & Y stage: 2.7 ± 0.5) who underwent PET, motor feature severity assessment using the Movement Disorder Society revised UPDRS, and the Dementia Rating Scale (DRS). Linear regression (R²_adj = 0.147; F_4,39 = 2.85; P = 0.036) showed that increased PIGD feature severity was associated with increased neocortical [¹¹C]‐Pittsburgh Compound B binding (β = 0.346; t₃₉ = 2.13; P = 0.039) while controlling for striatal [¹¹C]‐dihydrotetrabenazine binding, age, and DRS total score. Increased neocortical β‐amyloid deposition, even at low‐range levels, is associated with higher PIGD feature severity in PD patients at risk for dementia. This finding may explain why the PIGD motor phenotype is a risk factor for the development of PDD. © 2012 Movement Disorder Society     

NMDA receptor–dependent signaling pathways that underlie amyloid β‐protein disruption of LTP in the hippocampus

Alzheimer's disease (AD), the most common neurodegenerative disease in the elderly population, is characterized by the hippocampal deposition of fibrils formed by amyloid β‐protein (Aβ), a 40‐ to 42‐amino‐acid peptide. The folding of Aβ into neurotoxic oligomeric, protofibrillar, and fibrillar assemblies is believed to mediate the key pathologic event in AD. The hippocampus is especially susceptible in AD and early degenerative symptoms include significant deficits in the performance of hippocampal‐dependent cognitive abilities such as spatial learning and memory. Transgenic mouse models of AD that express C‐terminal segments or mutant variants of amyloid precursor protein, the protein from which Aβ is derived, exhibit age‐dependent spatial memory impairment and attenuated long‐term potentiation (LTP) in the hippocampal CA1 and dentate gyrus (DG) regions. Recent experimental evidence suggests that Aβ disturbs N‐methyl‐D ‐aspartic acid (NMDA) receptor–dependent LTP induction in the CA1 and DG both in vivo and in vitro. Furthermore, these studies suggest that Aβ specifically interferes with several major signaling pathways downstream of the NMDA receptor, including the Ca²⁺‐dependent protein phosphatase calcineurin, Ca²⁺/calmodulin‐dependent protein kinase II (CaMKII), protein phosphatase 1, and cAMP response element–binding protein (CREB). The influence of Aβ on each of these downstream effectors of the NMDA receptor is reviewed in this article. Additionally, other mechanisms of LTP modulation, such as Aβ attenuation of α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid (AMPA) receptor currents, are briefly discussed. © 2009 Wiley‐Liss, Inc.     

Twofold overexpression of human β-amyloid precursor proteins in transgenic mice does not affect the neuromotor,cognitive, or neurodegenerative sequelae following experimental brain injury

By using transgenic mice that overexpress human β-amyloid precursor proteins (APPs) at levels twofold higher than endogenous APPs, following introduction of the human APP gene in a yeast artificial chromosome (YAC), we examined the effects of controlled cortical impact (CCI) brain injury on neuromotor/cognitive dysfunction and the development of Alzheimer's disease (AD)-like neuropathology. Neuropathological analyses included Nissl-staining and immunohistochemistry to detect APPs, β-amyloid (Aβ), neurofilament proteins, and glial fibrillary acidic protein, whereas Aβ levels were measured in brain homogenates from mice subjected to CCI and control mice by using a sensitive sandwich enzyme-linked immunosorbent assay. Twenty APP-YAC transgenic mice and 17 wild type (WT) littermate controls were anesthetized and subjected to CCI (velocity, 5 m/second; deformation depth, 1 mm). Sham (anesthetized but uninjured) controls (n = 10 APP-YAC; n = 8 WT) also were studied. Motor function was evaluated by using rotarod, inclined-plane, and forelimb/hindlimb flexion tests. The Morris water maze was used to assess memory. Although CCI induced significant motor dysfunction and cognitive deficits, no differences were observed between brain-injured APP-YAC mice and WT mice at 24 hours and 1 week postinjury. By 1 week postinjury, both cortical and hippocampal CA3 neuron loss as well as extensive astrogliosis were observed in all injured animals, suggesting that overexpression of human APPs exhibited no neuroprotective effects. Although AD-like pathology (including amyloid plaques) was not observed in either sham or brain-injured animals, a significant decrease in brain concentrations of only Aβ terminating at amino acid 40 (Aβx-40) was observed following brain injury in APP-YAC mice (P < 0.05 compared with sham control levels). Our data show that the APP-YAC mice do not develop AD-like neuropathology following traumatic brain injury. This may be because this injury does not induce elevated levels of the more amyloidogenic forms of human Aβ (i.e., Aβx-42/43) in these mice. J. Comp. Neurol. 392:428–438, 1998. © 1998 Wiley-Liss, Inc.