A complex associative structure formed in the mammalian brain during acquisition of a simple visual discrimination task: dorsolateral striatum, amygdala, and hippocampus

A review of empirical evidence supporting the multiple memory systems view of the organization of learning and memory in the mammalian brain is presented as a powerful component of a broader foundation of basic scientific information necessary for understanding human behavior. However, it is argued that there are significant gaps in our knowledge about these different learning and memory systems, how they interact with one another, and how they interact with the rest of the brain. To demonstrate how little we know about these complex processes, this article reviews recent evidence showing the complexity of associative structure formed during the acquisition of a simple visual discrimination task. The results show that the dorsolateral striatum is necessary for the acquisition of this task but that both the amygdala and hippocampus incidentally acquire and store information during this training period. A new experiment is also presented showing that rats with complete or partial (dorsal vs. ventral) hippocampal lesions show a retrograde amnesic effect on the simple visual discrimination task despite the fact that these same lesions produce no impairment in the anterograde direction. Evidence is presented in support of one interpretation of this effect suggesting that the retrograde amnesia occurs, at least in part, because the hippocampus acquires a context-specific inhibitory association during original training. Although this representation is not required for acquisition of the task in the anterograde direction, removal of this representation has a disruptive effect on expression of the task.     

Did natural selection for increased cognitive ability in humans lead to an elevated risk of cancer?

Despite the overall genetic similarity that exists between humans and chimpanzees, the species are phenotypically distinct. Among the most notable distinctions are differences in brain size and cognitive abilities. Previous studies have shown that significant differences in gene expression exist between the human and chimpanzee brain. Integration of currently available gene expression data with known metabolic and signaling pathways indicates that the expression of genes involved in the programmed cell death of brain neurons is significantly different between humans and chimpanzees and predictive of a reduced level of neuron apoptosis in the human brain. This pattern of expression is generally maintained in other human organs suggesting that apoptosis is reduced in humans relative to chimpanzees. We propose that a decreased rate of programmed neuron death may have been a consequence of selection for increased cognitive ability in humans. Since reduced apoptotic function is associated with an increased risk of cancer and related diseases, we hypothesize that selection for increased cognitive ability in humans coincidently resulted in an increased risk of cancer and other diseases associated with reduced apoptotic function.     

Assessment of microvessel density and carbonic anhydrase-9 (CA-9) expression in rectal cancer

Aim

The mechanism by which neoplasias respond to hypoxia determines their biological behavior and prognosis. Understanding the biology of tumors under hypoxic conditions is crucial for the development of anti-angiogenic therapy. Using the largest cohort of rectal adenocarcinomas to date, this study aimed to assess microvessel density (MVD) and carbonic anhydrase-9 (CA-9) expression and to correlate the results with recurrence and cancer-specific survival.

Materials and methods

Patients (n=101) who underwent surgery for rectal adenocarcinoma without previous neoadjuvant therapy or metastatic disease were selected. MVD and CA-9 expression were assessed immunohistologically by using the CD34 antibody and the MN/CA9 M75 antibody, respectively. In a multifactorial analysis, the results were correlated with tumor stage, recurrence rate, and long-term survival.

Results

MVD was higher with increased T- and N-stages (p<0.01) and associated positively with poor survival (hazard ratio (HR) 1.3 per 10 vessel increase, p<0.01). CA-9 was expressed in 73% of cancers. Negative lymph node status correlated with CA-9 positivity (p<0.05), reflected in a higher rate of CA-9 positivity in earlier Dukes’ stages (p<0.05). CA-9 positivity across tumor node metastasis (TNM) stages approached significance (Stage I/II: 80% CA-9 positive vs. 20% CA-9 negative; Stage III: 63% CA-9 positive vs. 37% negative, p=0.051). A trend was seen towards better cancer-specific survival in patients with CA-9 positive carcinomas (HR 0.51, p=0.07) on univariate analysis.

Discussion

MVD was higher in more advanced T- and N-stages and may be used as a determinant of survival in patients with rectal adenocarcinomas. CA-9 expression was seen more often in earlier Dukes’ stages, possibly representing an early tumor hypoxic response. CA-9 expression by adenocarcinoma cells may confer long-term survival advantage in surgically treated rectal cancer.     

Elimination of GD3 synthase improves memory and reduces amyloid-beta plaque load in transgenic mice

Gangliosides have been shown to be necessary for beta-amyloid (Abeta) binding and aggregation. GD3 synthase (GD3S) is responsible for biosynthesis of the b- and c-series gangliosides, including two of the four major brain gangliosides. We examined Abeta-ganglioside interactions in neural tissue from mice lacking the gene coding for GD3S (St8sia1), and in a double-transgenic (APP/PSEN1) mouse model of Alzheimer's disease cross-bred with GD3S-/- mice. In primary neurons and astrocytes lacking GD3S, Abeta-induced cell death and Abeta aggregation were inhibited. Like GD3S-/- and APP/PSEN1 double-transgenic mice, APP/PSEN1/GD3S-/- "triple-mutant" mice are indistinguishable from wild-type mice on casual examination. APP/PSEN1 double-transgenics exhibit robust impairments on a number of reference-memory tasks. In contrast, APP/PSEN1/GD3S-/- triple-mutant mice performed as well as wild-type control and GD3S-/- mice. Consistent with the behavioral improvements, both aggregated and unaggregated Abeta and associated neuropathology were almost completely eliminated in triple-mutant mice. These results suggest that GD3 synthase may be a novel therapeutic target to combat the cognitive deficits, amyloid plaque formation, and neurodegeneration that afflict Alzheimer's patients.     

Regulation of wild-type and mutant KCNQ1/KCNE1 channels by tyrosine kinase

The objective of the study was to investigate the role of tyrosine phosphorylation in the regulation of KCNQ1/KCNE1 channels. Large whole-cell time- and voltage-dependent K⁺ currents were present in human embryonic kidney 293 cells cotransfected with human KCNQ1 and KCNE1 but not in control nontransfected cells. The time- and voltage-dependent current had biophysical properties typical of cardiac KCNQ1/KCNE1 current and was almost completely abolished by KCNQ1 blocker chromanol 293B (50 μM). Both KCNQ1/KCNE1 and KCNQ1 current were inhibited in a voltage-independent manner by tyrosine kinase (PTK) inhibitor tyrphostin A25 (100 μM), but not by PTK-inactive tyrphostin A1 (100 μM), suggesting involvement of tyrosine phosphorylation in maintaining channel activity. This view was strengthened by the finding that phosphotyrosyl phosphatase inhibitor monoperoxo(picolinato)-oxo-vanadate(V) (200 μM) reversed the inhibition of current by tyrphostin A25. However, the channel-pertinent tyrosine phosphorylation modulated by these compounds does not appear to be on the channel itself because inhibition of current by tyrphostin A25 was unaffected by single and multiple mutations of KCNQ1 cytoplasmically accessible tyrosine residues. Inhibition by tyrphostin A25 was unaffected by intracellularly applied diC8 phosphatidylinositol-4,5-bisphosphate (diC8 PIP₂; 25 μM), and based on the results obtained from cell surface biotinylation experiments, it was not due to loss of channels from the membrane. We conclude that tyrphostin A25 inhibits KCNQ1/KCNE1 current by lowering tyrosine phosphorylation on unidentified nonchannel protein(s) that directly or indirectly regulate the open probability of the KCNQ1 pore in a PIP₂-independent manner.     

Tracking the voluntary control of auditory spatial attention with event-related brain potentials

A lateralized event-related potential (ERP) component elicited by attention-directing cues (ADAN) has been linked to frontal-lobe control but is often absent when spatial attention is deployed in the auditory modality. Here, we tested the hypothesis that ERP activity associated with frontal-lobe control of auditory spatial attention is distributed bilaterally by comparing ERPs elicited by attention-directing cues and neutral cues in a unimodal auditory task. This revealed an initial ERP positivity over the anterior scalp and a later ERP negativity over the parietal scalp. Distributed source analysis indicated that the anterior positivity was generated primarily in bilateral prefrontal cortices, whereas the more posterior negativity was generated in parietal and temporal cortices. The anterior ERP positivity likely reflects frontal-lobe attentional control, whereas the subsequent ERP negativity likely reflects anticipatory biasing of activity in auditory cortex.     

Contrasting Actions of Selective Inhibitors of Angiopoietin-1 and Angiopoietin-2 on the Normalization of Tumor Blood Vessels

Angiopoietin-1 (Ang1) and angiopoietin-2 (Ang2) have complex actions in angiogenesis and vascular remodeling due to their effects on Tie2 receptor signaling. Ang2 blocks Ang1-mediated activation of Tie2 in endothelial cells under certain conditions but is a Tie2 receptor agonist in others. We examined the effects of selective inhibitors of Ang1 (mL4-3) or Ang2 (L1-7[N]), alone or in combination, on the vasculature of human Colo205 tumors in mice. The Ang2 inhibitor decreased the overall abundance of tumor blood vessels by reducing tumor growth and keeping vascular density constant. After inhibition of Ang2, tumor vessels had many features of normal blood vessels (normalization), as evidenced by junctional accumulation of vascular endothelial-cadherin, junctional adhesion molecule-A, and platelet/endothelial cell adhesion molecule-1 in endothelial cells, increased pericyte coverage, reduced endothelial sprouting, and remodeling into smaller, more uniform vessels. The Ang1 inhibitor by itself had little noticeable effect on the tumor vasculature. However, when administered with the Ang2 inhibitor, the Ang1 inhibitor prevented tumor vessel normalization, but not the reduction in tumor vascularity produced by the Ang2 inhibitor. These findings are consistent with a model whereby inhibition of Ang2 leads to normalization of tumor blood vessels by permitting the unopposed action of Ang1, but decreases tumor vascularity primarily by blocking Ang2 actions.Solid tumors require angiogenesis—the formation of new blood vessels from existing vessels—for survival, growth, and metastasis.¹ Tumor vessels are structurally and functionally abnormal.^1,2 They exist in a constantly dynamic state of sprout formation, proliferation, remodeling, or regression. Structurally, tumor vessels tend to be leaky and tortuous, lacking the hierarchical arrangement of arterioles, capillaries, and venules.² Pericytes that attach to and help stabilize normal vessels are loosely associated with the endothelium of tumor vessels.^1,2 These vascular abnormalities result in impaired and heterogeneous blood flow. In tumors, angiogenesis inhibitors not only cause vessel regression or retardation of vessel growth, but they can also induce vascular normalization.^1,2,3The complicated regulation of angiogenesis and vascular maturation involves multiple signaling cascades driven by endothelial-cell specific growth factors and their receptors. One of these, vascular endothelial growth factor (VEGF) has been extensively studied,⁴ but angiopoietins and other growth factors are also involved.^5,6 The angiopoietin ligands (Ang1 and Ang2) and their receptor (Tie2) have essential roles in vascular development.^7,8 Ang1 is produced by vascular mural cells, pericytes, and certain other cells, whereas Ang2 and Tie2 are expressed primarily by endothelial cells.Angiogenesis and vascular remodeling involve a complex coordination of Ang1 and Ang2 signaling through Tie2.⁵ The traditional view of Ang1 and Ang2 signaling is that the growth factors have opposing effects on Tie2 receptor activation: Ang1 binds to Tie2 to promote vascular maturation and integrity, whereas Ang2 acts as a naturally occurring antagonist of Ang1.^7,8,9,10,11 Although a number of studies indicate an antagonistic role of Ang2, recent studies have shown that Ang2 can have an agonistic role depending on the experimental environment.^12,13,14,15 If expressed at high concentrations or for long durations in cultured endothelial cells, Ang2—like Ang1—can induce Tie2 receptor phosphorylation.^13,16 Ang2 can also promote chemotaxis, tube formation, migration, and sprouting of endothelial cells in the absence of Ang1,¹⁴ which support the view that Ang2 actions are context- dependent.Normalization of tumor vascular morphology and function has been demonstrated with numerous angiogenesis inhibitors.^1,17,18 Ang1 and Ang2 regulate vascular maturation and integrity during development; however, their effects on normalization of tumor vessels are not known. Tumors grown in mice lacking Ang2 have a more mature vascular phenotype, but it is not known whether Ang1 plays a role.¹⁹ The effects of individual angiopoietins on the tumor vasculature have not been studied extensively in loss-of-function experiments, due largely to the limited availability of selective angiopoietin inhibitors. Some clues to the effects of Ang1 and Ang2 on tumor vessels have been garnered through overexpression of the ligands in tumor cell xenografts.^{20,21,22,23,24,25,26} These studies, however, have yielded conflicting data,^{20,21,22,23,24,25,26} the ligands were administered at nonphysiological levels, and the results were restricted to prevention studies. Studies blocking the Tie2 receptor have shown reduced tumor angiogenesis,^27,28,29,30 but the specific roles of each ligand cannot be differentiated. Pharmacological angiopoietin inhibitors using antisense, aptamer, and peptide-Fc fusion protein (peptibody) technologies are currently being developed, but published studies have been restricted to inhibition of Ang1 or Ang2 alone.^31,32,33 Studies using aptamers or peptibodies that potently neutralize Ang2 activity showed that Ang2 antagonism resulted in inhibition of angiogenesis and tumor growth.^31,32 Inhibition of Ang1 in a cell line stably transfected with antisense RNA resulted in reduced tumor growth and angiogenesis.³³To gain a better understanding of the effects of Ang1 and Ang2 on blood vessels in tumors, we used selective inhibitors (peptibodies) of Ang1 and Ang2, alone or in combination, in Colo205 tumors. These studies focused on Colo205 tumors, as this model is sensitive to angiopoietin inhibitors.³¹ We found that inhibition of Ang1 alone had little effect on the tumor vasculature, whereas inhibition of Ang2 resulted in fewer tumor vessels and normalization of the surviving tumor vessels. When the Ang2 inhibitor was administered with the Ang1 inhibitor, tumor vessel normalization did not occur, but the Ang2 inhibitor-mediated reduction in vascularity was unaffected. These findings suggest that inhibition of Ang2 leads to unopposed Ang1 activity and results in normalization of tumor vessels. In contrast, the Ang2 inhibitor-mediated reduction in tumor vascularity was Ang1-independent.     

Anti‐retroviral effects of type I IFN subtypes in vivo

Type I IFN play a very important role in immunity against viral infections. Murine type I IFN belongs to a multigene family including 14 IFN‐α subtypes but the biological functions of IFN‐α subtypes in retroviral infections are unknown. We have used the Friend retrovirus model to determine the anti‐viral effects of IFN‐α subtypes in vitro and in vivo. IFN‐α subtypes α1, α4, α6 or α9 suppressed Friend virus (FV) replication in vitro, but differed greatly in their anti‐viral efficacy in vivo. Treatment of FV‐infected mice with the IFN‐α subtypes α1, α4 or α9, but not α6 led to a significant reduction in viral loads. Decreased splenic viral load after IFN‐α1 treatment correlated with an expansion of activated FV‐specific CD8⁺ T cells and NK cells into the spleen, whereas in IFN‐α4‐ and ‐α9‐treated mice it exclusively correlated with the activation of NK cells. The results demonstrate the distinct anti‐retroviral effects of different IFN‐α subtypes, which may be relevant for new therapeutic approaches.     

Neutralizing Monoclonal Antibodies Directed against Defined Linear Epitopes on Domain 4 of Anthrax Protective Antigen

The anthrax protective antigen (PA) is the receptor-binding subunit common to lethal toxin (LT) and edema toxin (ET), which are responsible for the high mortality rates associated with inhalational Bacillus anthracis infection. Although recombinant PA (rPA) is likely to be an important constituent of any future anthrax vaccine, evaluation of the efficacies of the various candidate rPA vaccines is currently difficult, because the specific B-cell epitopes involved in toxin neutralization have not been completely defined. In this study, we describe the identification and characterization of two murine monoclonal immunoglobulin G1 antibodies (MAbs), 1-F1 and 2-B12, which recognize distinct linear neutralizing epitopes on domain 4 of PA. 1-F1 recognized a 12-mer peptide corresponding to residues 692 to 703; this epitope maps to a region of domain 4 known to interact with the anthrax toxin receptor CMG-2 and within a conformation-dependent epitope recognized by the well-characterized neutralizing MAb 14B7. As expected, 1-F1 blocked PA''s ability to associate with CMG-2 in an in vitro solid-phase binding assay, and it protected murine macrophage cells from intoxication with LT. 2-B12 recognized a 12-mer peptide corresponding to residues 716 to 727, an epitope located immediately adjacent to the core 14B7 binding site and a stretch of amino acids not previously identified as a target of neutralizing antibodies. 2-B12 was as effective as 1-F1 in neutralizing LT in vitro, although it only partially inhibited PA binding to its receptor. Mice passively administered 1-F1 or 2-B12 were partially protected against a lethal challenge with LT. These results advance our fundamental understanding of the mechanisms by which antibodies neutralize anthrax toxin and may have future application in the evaluation of candidate rPA vaccines.Bacillus anthracis has long been recognized as a serious public health threat, given the ease with which B. anthracis spores can be disseminated via aerosol and due to the high mortality rate that accompanies spore inhalation. These fears were realized in the fall of 2001, when B. anthracis spores were circulated through the U.S. postal system, resulting in five deaths, 22 known cases of infection, and the possible exposure of more than 30,000 people (7). Although routine vaccination of civilians against anthrax is neither necessary nor desirable, certain segments of the population, notably emergency first responders and research laboratory personnel, remain at risk of exposure and are in need of an effective preexposure vaccine. In the United States, the only licensed anthrax vaccine, Anthrax Vaccine Adsorbed (AVA) or Biothrax, has been subject to controversy for years, due to safety concerns (3), and is not considered an ideal vaccine due to its protracted vaccination schedule (six injections over 18 months). AVA, which consists of formalin-treated culture filtrate from an attenuated strain of B. anthracis adsorbed to aluminum hydroxide, is also inherently difficult to manufacture and to standardize. A recombinant anthrax vaccine, manufactured by VaxGen, was considered to be a leading candidate to replace AVA and to supply the Strategic National Stockpile with 75 million doses, but it was recently discontinued because of concerns regarding vaccine production and stability. For these reasons, there is now a concerted effort to develop a new anthrax vaccine based on a defined antigenic composition.Current anthrax vaccines are focused on protective antigen (PA), the primary determinant in AVA that is involved in immunity to anthrax infection (21). PA is the receptor-binding subunit common to both lethal factor (LF), and edema factor (EF), which, upon PA binding form lethal toxin (LT) and edema toxin (ET), respectively. The anthrax toxins are responsible for the high mortality rates associated with inhalational B. anthracis infection. PA is secreted by B. anthracis as an 83-kDa protein (PA₈₃) that binds two known anthrax toxin receptors (ATRs), namely, tumor endothelium marker 8 (TEM-8) and capillary morphogenesis protein 2 (CMG-2) (5, 6, 19). Following receptor engagement, the amino-terminal 20-kDa region of the PA (PA₂₀) is proteolytically cleaved by a furinlike protease, exposing a region of PA that allows for the formation of the holotoxin. The membrane-bound 63-kDa PA (PA₆₃) spontaneously oligomerizes into heptamers and then associates either with LF, a zinc-dependent protease specific for certain members of the mitogen-activated protein kinase kinase family (12, 13), or EF, a calmodulin-, Ca²⁺-dependent adenylate cyclase (20), to form LT and ET.It is well established that animals immunized with recombinant PA, or passively administered anti-PA antiserum, are protected against LT/ET exposure or spore challenge (reviewed in references 16 and 7). For example, we recently reported that LT-challenged mice treated with goat anti-PA antiserum (either alone or in combination with antibiotics) demonstrated increased survival rates compared to untreated mice (18). However, the antibody response to PA is complicated: total anti-PA titers, as measured by enzyme-linked immunosorbent assays (ELISAs), do not correlate well with protection (35). This is likely due to the fact that a large number of anti-PA antibodies are directed against non-neutralizing (or possibly even toxin-enhancing) epitopes (29, 41). Pioneering work by Little et al. (23) suggests that at least 20 antigenic determinants exist on PA. At present, only a few of these epitopes has been described in detail; several have been shown to be targets of monoclonal antibodies (MAbs) capable of effective neutralization in vivo or in vitro (1, 8, 10, 17, 44, 45). The fact that key neutralizing epitopes on PA remain widely unidentified poses a significant barrier to the evaluation of the efficacy of vaccines based on full-length and truncated PA subunits.PAs can be divided into four functional domains. Domain 1 (residues 1 to 258) contains the furin recognition site (¹⁶⁴RKKR¹⁶⁷), as well as the region of PA that is recognized by LF and EF (30). Domain 2 (residues 259 to 487) and part of domain 3 (residues 488 to 595) are implicated in heptamerization, pore formation, and translocation of EF/LF across endosomal membranes (4, 27, 28, 34). Domain 4 (residues 596 to 735) constitutes the region of the PA involved in receptor recognition and attachment (39). Antibodies against domain 4 are postulated to be the most effective in neutralizing LT and ET (1), since they are proposed to interfere with PA binding to ATR. However, the only neutralizing epitope that has been characterized in detail is the one recognized by MAb 14B7 (24, 37, 40). This MAb contacts the face of domain 4 that is involved in receptor recognition, and has been proposed to encompass residues 671 to 721. Certainly, additional neutralizing epitopes exist on domain 4 (1, 46). In particular, Abboud and Casadevall (1) suggested a linear epitope, immediately adjacent to or possibly overlapping the 14B7 binding site, as the target of neutralizing antibodies. The characterization of antibodies that are both linear epitope dependent and neutralizing will be useful in the evaluation specific correlates of immunity; such MAbs can be incorporated into functional screening assays of potential vaccine candidates.The goal of our study was to identify additional B-cell epitopes, within domain 4 of PA, that constitute the targets of neutralizing MAbs. Toward this end, we screened a collection of B-cell hybridomas, produced from PA₈₃-immunized mice, for MAbs capable of binding to a domain 4-specific peptide array. We identified two domain 4-reactive MAbs, 1-F1 and 2-B12. 1-F1 recognized a peptide spanning residues 692 to 703; this epitope maps to a region of domain 4 that overlaps with the binding site recognized by the well-characterized neutralizing antibody 14B7 (residues 671 to 721) (23, 40). 2-B12, on the other hand, recognized a peptide spanning residues 716 to 727; this epitope maps adjacent to the core 14B7 binding site and at a location not previously identified as being a target of neutralizing antibodies. Both MAbs neutralized LT in vitro and partially protected mice against LT challenge. These results advance our fundamental understanding of the mechanisms by which antibodies neutralize anthrax toxin and may have future application in the evaluation of candidate rPA vaccines.