Epstein-Barr virus-associated inflammatory pseudotumor variant of follicular dendritic cell sarcoma of the liver: a case report and review of the literature.

BACKGROUND: Follicular dendritic cell sarcoma is a rare stromal tumor with no standard treatment. However, some reports have revealed that follicular dendritic cell sarcoma has an inflammatory pseudotumor variant associated with Epstein-Barr virus infection that has a relatively good prognosis. In this report, we present a case of a resected inflammatory pseudotumor variant of follicular dendritic cell sarcoma of the liver, and have reviewed the literature on the clinicopathological, molecular, and genomic features of this tumor. CASE PRESENTATION: The inflammatory pseudotumor variant of follicular dendritic cell sarcoma originates only in the liver or spleen, causes no symptoms, and is more common in middle-aged Asian women. It has no characteristic imaging features, which partially explains why the inflammatory pseudotumor variant of follicular dendritic cell sarcoma is difficult to diagnose. Pathologically, the inflammatory pseudotumor variant of follicular dendritic cell sarcoma has spindle cells mixed with inflammatory cells and is variably positive for follicular dendritic cell markers (CD21, CD23, and CD35) and Epstein-Barr virus-encoded RNA. On genetic analysis, patients with this tumor high levels of latent membrane protein 1 gene expression and extremely low levels of host C-X-C Chemokine Receptor type 7 gene expression, indicating that the inflammatory pseudotumor variant of follicular dendritic cell sarcoma has a latent Epstein-Barr virus type 2 infection. CONCLUSIONS: The inflammatory pseudotumor variant of follicular dendritic cell sarcoma is an Epstein-Barr virus-associated tumor and a favorable prognosis by surgical resection, similar to Epstein-Barr virus-associated gastric cancer.

Prognostic significance of overexpression of c-Met oncoprotein in cholangiocarcinoma.

BACKGROUND: Cholangiocarcinoma (CC) is a highly malignant carcinoma. We attempted to clarify the prognostic significance of c-Met overexpression and its association with clinicopathological factors in patients with CC. PATIENTS AND METHODS: One hundred and eleven patients with intrahepatic CC (IHCC) and 136 patients with extrahepatic CC (EHCC) who had undergone curative surgery were divided immunohistologically into c-Met(high) and c-Met(low) groups. Clinicopathological factors and outcomes were compared between the groups. c-Met and epidermal growth factor receptor (EGFR) expression was also examined in 10 CC cell lines. RESULTS: The positivity of c-Met was 45.0% in IHCC and 68.4% in EHCC; c-Met(high) expression was demonstrated in 11.7% of IHCC and 16.2% of EHCC. c-Met(high) expression was significantly correlated with the 5-year survival rate for CC overall (P=0.0046) and for IHCC (P=0.0013), histopathological classification in EHCC, and for EGFR overexpression in both IHCC and EHCC. Coexpression and coactivation of c-Met and EGFR were also observed in CC cell lines. Multivariate analysis revealed that c-Met(high) expression was an independent predictor of poor overall and disease-free survival in patients with IHCC. CONCLUSIONS: c-Met overexpression is associated with EGFR expression and is a poor prognostic factor in CC.

Phase II study of trifluridine/tipiracil plus bevacizumab by RAS mutation status in patients with metastatic colorectal cancer refractory to standard therapies: JFMC51-1702-C7.

BACKGROUND: Although the efficacy of trifluridine/tipiracil (FTD/TPI) plus bevacizumab (BEV) against metastatic colorectal cancer (mCRC) has been demonstrated, little is known about its effectiveness upon disease stratification by RAS mutations. In this phase II study, we investigated the efficacy and safety profiles of FTD/TPI in mCRC according to RAS mutation status. PATIENTS AND METHODS: Eligible patients were mCRC refractory or intolerant to all standard therapies other than FTD/TPI and regorafenib. Patients received 4-week cycles of treatment with FTD/TPI (35 mg/m2, twice daily, days 1-5 and 8-12) and bevacizumab (5 mg/kg, days 1 and 15). The primary endpoint was disease control rate (DCR). The null hypothesis of DCR in both RAS wild-type (WT) and mutant (MUT) cohorts was 44%, assuming a one-sided significance level of 5.0%. The necessary sample size was estimated to be 49 patients (target sample size: 50 patients) for each cohort. RESULTS: Between January and September 2018, 102 patients were enrolled, and 97 patients fulfilled the eligibility criteria (48 in the RAS WT cohort and 49 in the RAS MUT cohort). DCRs in the RAS WT and MUT cohort were 66.7% [90% confidence interval (CI), 53.9%-77.8%, P = 0.0013] and 55.1% (90% CI, 42.4%-67.3%, P = 0.0780), respectively. The median progression-free survival (PFS) and overall survival (OS) were 3.8 and 9.3 months, respectively, in the RAS WT cohort and 3.5 and 8.4 months, respectively, in the RAS MUT cohort. The most common grade 3 or higher adverse event in both cohorts was neutropenia (46% in the RAS WT cohort and 62% in the RAS MUT cohort), without unexpected safety signals. CONCLUSIONS: FTD/TPI plus bevacizumab showed promising activity with an acceptable safety profile for pretreated mCRC, regardless of RAS mutation status, although the efficacy outcomes tended to be better in RAS WT.

Clinical impact of intraoperative histological examination of the ductal resection margin in extrahepatic cholangiocarcinoma.

BACKGROUND: Although ductal resection margin status in extrahepatic cholangiocarcinoma is evaluated by intraoperative histological examination of frozen sections, its clinical relevance remains controversial. METHODS: Material taken from patients who underwent R0 or R1 resection for extrahepatic cholangiocarcinoma with intraoperative histological examination of the final ductal resection margins between 1994 and 2003 were reviewed. The following histological classification was used: insufficient, negative for malignancy (NM), undetermined lesion (UDL) or positive for malignancy (PM). Multivariable analyses of overall survival and anastomotic recurrence in relation to ductal margin status were performed. RESULTS: Resection material from 363 patients was identified. For the proximal ductal margin, only PM in intramural lesions was significantly associated with poor survival (hazard ratio (HR) 1.72, 95 per cent confidence interval (c.i.) 1.06 to 2.74) and anastomotic recurrence (HR 6.39, 95 per cent c.i. 1.89 to 21.62) compared with NM. In analysis of overall survival according to distal ductal margin status, the HRs for UDL and PM lesions in comparison with NM were not significant. CONCLUSION: PM in intramural lesions found during intraoperative histological examination of the proximal ductal resection margin was related to clinical outcome. This finding favours additional resection of the bile duct. A similar association was not found for histology results of the distal resection margin.

Vandetanib (ZD6474), an inhibitor of VEGFR and EGFR signalling, as a novel molecular-targeted therapy against cholangiocarcinoma.

Cholangiocarcinoma is an intractable cancer, with no effective therapy other than surgical resection. Elevated vascular endothelial growth factor (VEGF) and epidermal growth factor receptor (EGFR) expressions are associated with the progression of cholangiocarcinoma. We therefore examined whether inhibition of VEGFR and EGFR could be a potential therapeutic target for cholangiocarcinoma. Vandetanib (ZD6474, ZACTIMA), a VEGFR-2/EGFR inhibitor, was evaluated. Four human cholangiocarcinoma cell lines were molecularly characterised and investigated for their response to vandetanib. In vitro, two cell lines (OZ and HuCCT1), both of which harboured KRAS mutation, were refractory to vandetanib, one cell line (TGBC24TKB) was somewhat resistant, and another cell line (TKKK) was sensitive. The most sensitive cell line (TKKK) had EGFR amplification. Vandetanib significantly inhibited the growth of TKKK xenografts at doses > or = 12.5 mg kg(-1) day(-1) (P<0.05), but higher doses (50 mg kg(-1) day(-1), P<0.05) of vandetanib were required to inhibit the growth of OZ xenografts. Vandetanib (25 mg kg(-1) day(-1)) also significantly (P=0.006) prolonged the time to metastasis in an intravenous model of TKKK metastasis. Inhibiting both VEGFR and EGFR signalling appears a promising therapeutic approach for cholangiocarcinoma. The absence of KRAS mutation and the presence of EGFR amplification may be potential predictive molecular marker of sensitivity to EGFR-targeted therapy in cholangiocarcinoma.

Microtopography of the dual corticothalamic projections originating from domains along the frequency axis of the cat primary auditory cortex.

Spatial relationships between clusters of corticothalamic (CT) large terminals originating from cortical domains tuned to different frequencies were examined by pair-injecting two different anterograde tracers. Large-terminal CT projection originating from layer 5 was highly divergent with each injection site producing, on average, 15 local clusters distributing throughout non-lemniscal thalamic nuclei following a single anterograde tracer injection in the cat primary auditory cortex. Paired injections in higher- and lower-frequency cortical domains, resulting in labeling of two independent sets of terminal clusters, showed five recognizable patterns of spatial interaction between them. (1) In the ventral division of the medial geniculate complex (vMGC), sheet-like plexuses of small terminals of different origins were situated in parallel, with minimal overlap. (2) Extensive overlap of two low-density plexuses of differently labeled small terminals was observed in the medial division of the medial geniculate complex (MGC). (3) At the transition zones between the vMGC and the superficial dorsal nucleus of the MGC dorsal division, and between the vMGC and the ventrolateral nucleus, there were relatively broad clusters of a high density of large-terminal structures from the two cortical domains, which overlapped extensively. (4) At multiple loci in the nonlemniscal nuclei, pairing of two small clusters of differently labeled large terminals was observed. (5) Small unpaired clusters of large terminals were also found in the nonlemniscal nuclei. For large terminals, approximately 14%, 59%, and 27% clusters per injection demonstrated patterns 3, 4, and 5, respectively. The results provide evidence for the precise topographical organization for the large-terminal CT system at the microscopic level despite its highly divergent projection. This microtopographical projection from the tonotopic cortical field to non-tonotopic thalamic nuclei may raise the possibility of presence of a map that has not been defined in auditory non-lemniscal thalamic nuclei yet.

Extramedullary myeloid tumour (EMMT) of the gallbladder.

This report describes a rare case of an extramedullary myeloid tumour (EMMT) of the gallbladder in a patient without leukaemia. A 33 year old man visited a local hospital because of jaundice. Abdominal computed tomography revealed a tumorous mass measuring 6.0 x 4.5 cm and involving the entire gallbladder. A percutaneous needle biopsy was attempted, but because adenocarcinoma could not be completely ruled out, the use of undue force was considered dangerous. Under a preoperative diagnosis of gallbladder carcinoma, a hepatopancreatoduodenectomy was performed. The tumour cells exhibited various amounts of eosinophilic cytoplasm, had medium sized round nuclei with indentation and grooving, and were strongly immunoreactive for myeloperoxidase, CD43, and c-kit protein (CD117). After surgery, the patient underwent combination chemotherapy as prescribed for cases of acute myeloblastic leukaemia. The patient did not develop acute leukaemia during a follow up period of four years. In conclusion, a correct diagnosis of EMMT can be made using appropriate immunohistochemical staining.

Model platinum nucleobase and nucleoside complexes and antitumor activity: X-ray crystal structure of [PtIV(trans-1R,2R-diaminocyclohexane)trans-(acetate)2(9-ethylguanine)Cl]NO3.H2O.

A series of platinum(II) and (IV) monoadducts of the type [Pt(II)(DACH)LCl]NO3 and [Pt(IV)(DACH)trans-(X)2LCl]NO3 (where DACH=trans-1R,2R-diaminocyclohexane, L=adenine, guanine, hypoxanthine, cytosine, adenosine, guanosine, inosine, cytidine, 9-ethylguanine (9-EtGua), or 1-methylcytosine and X=hydroxo or acetato ligand) have been synthesized and characterized by elemental analysis and by 1H and 195Pt nuclear magnetic resonance (NMR) spectroscopy. The crystal structure of the model nucleobase complex [Pt(IV)(trans-1R,2R-diaminocyclohexane)trans-(acetate)2(9-EtGua)Cl]NO3.H2O was determined using a single crystal X-ray diffraction method. The compound crystallized in the monoclinic space group P2(1), with a=10.446(2) A, b=22.906(5) A, c=10.978(2) A, Z=4, and R=0.0718, based upon the total of 11,724 collected reflections. In this complex, platinum had a slightly distorted octahedron geometry owing to the presence of a geometrically strained five-member ring. The two adjacent corners of the platinum plane were occupied by the two amino nitrogen of DACH, whereas, the other two equatorial positions occupied by chloride ion and 9-ethylguanine. The remaining two axial positions were occupied by the oxygen atoms of acetato ligands. The DACH ring was in a chair configuration. An intricate network of intermolecular hydrogen bonds held the crystal lattice together. Some of these synthesized models of DACH-Pt-DNA adducts have good in vitro cytotoxic activity against the cisplatin-sensitive human cancer ovarian A2780 cell line (IC50=1-8 microM). Interestingly, a substituted nucleobase (9-ethylguanine) adduct was over 6-fold more potent than regular adducts. The cross-resistance factor against the 44-fold cisplatin-resistant 2780CP/clone 16 cells was about 3-9; thus, the cytotoxicity of adducts was indicative of low potency, but the resistance factors were also substantially low. These results suggest that DNA adducts of DACH-Pt are cytotoxic with low cross-resistance.

Labeling of pyramidal and nonpyramidal neurons with lectin Vicia villosa during postnatal development of the guinea pig.

Labeling of cortical neurons with a lectin, Vicia villosa (VVA), was investigated in guinea pigs aged 1 day old to adult. Lectin histochemistry revealed a perineuronal sheath, which outlined the cell bodies, apical dendrites, and axon initial segments, in distinct populations of pyramidal and nonpyramidal neurons. Their laminar positions were segregated, with the pyramidal neurons confined to layer 5 and the nonpyramidal neurons distributed mainly in layers 3-5. The VVA-labeled substance(s) was detected at the interfaces between neurons and cellular elements present in the perisynaptic region, including glial processes and fine axons. However, it was excluded from synaptic clefts of presynaptic terminals. Double immunohistochemistry revealed that most of the VVA-labeled neurons were also labeled perineuronally with a monoclonal antibody, Cat 301, and vice versa. Dendritic patterns of the VVA-labeled pyramidal neurons were studied further by intracellular injection of Lucifer yellow into fixed slices. Apical dendrites had a considerable thickness before arborizing into a few daughter branches in layer 3 or 4, suggesting a morphological resemblance to intrinsic, bursting pyramidal neurons defined physiologically in vitro. During postnatal development, there was a global spatiotemporal pattern in the onset of VVA labeling of the cortical neurons. The labeling progressed from medial and posterior cortical areas, which are closely related to the hippocampal formation, to more lateral and anterior areas, which are less closely related. The labeling patter thus tends to follow the order of the phylogenetical development of the isocortex.

Cholinergic innervation of the rat cerebellum: qualitative and quantitative analyses of elements immunoreactive to a monoclonal antibody against choline acetyltransferase.

Cholinergic innervation of the rat cerebellum was investigated immunohistochemically by using a monoclonal antibody against choline acetyltransferase. Immunoreactive structures included: 1) a subpopulation of mossy fibers and glomerular rosettes; 2) thin varicose fibers, which were closely associated with the Purkinje cell layer and also found in the molecular layer; and 3) relatively dense networks of varicose fibers distributing in the cerebellar nuclei. Quantitative analysis indicated that a great many immunoreactive rosettes were localized in lobules IXc and X, although their density in lobule X was approximately four times that in the lobule IXc. A considerable number of immunoreactive structures were also present in all other lobules. In the hemispheres they were confined to a zone immediately beneath the Purkinje cell layer, whereas in the vermis they were scattered throughout the granular layer. Most of the immunoreactive fibers found in the molecular layer coursed toward the pial surface and were distributed within the inner half of the molecular layer. In the cerebellar nuclei, portions of the medial nucleus and magnocellular portion of the lateral nucleus had moderately dense networks of immunoreactive fibers, whereas loose networks of fibers were observed in the posterior interposed nucleus. Other parts of the cerebellar nuclei contained a smaller number of varicose fibers.

Distribution of dendrites of mitral, displaced mitral, tufted, and granule cells in the rabbit olfactory bulb.

To determine the dendritic fields, mitral, displaced mitral, middle tufted, and granule cells in the rabbit olfactory bulb were stained by intracellular injection of HRP. The secondary dendrites of mitral cells were distributed mostly in the inner half of the external plexiform layer (EPL). Those of displaced mitral cells extended mainly into the middle and superficial sublayers in the EPL. The secondary dendrites of middle tufted cells were distributed mostly in the superficial portion of the EPL. Mitral cells extended their secondary dendrites in virtually all directions within a plane tangential to the mitral cell layer (MCL) and thus had a disklike projection field with a radius of about 850 microns. Displaced mitral cells had similar dendritic projection fields in the tangential plane but with somewhat distorted shapes. The secondary dendrites of middle tufted cells had a tendency to extend in particular directions. From the projection pattern of the gemmules on the peripheral processes, granule cells were classified into three types. Type I granule cells had gemmules both in the superficial and in the deep sublayers of the EPL. The peripheral processes of Type II granule cells were confined to the deep half of the EPL. The gemmules of Type III granule cells ere distributed in the superficial half of the EPL. The differing dendritic ramification among mitral, displaced mitral, and middle tufted cells suggests the separation of the dendrodendritic synaptic interactions with granule cells in different sublayers in the EPL. It also suggests a functional separation of the sublayers of the EPL.

The trajectory of mitral cell axons in the rabbit olfactory cortex revealed by intracellular HRP injection.

The projection of the axon and axon collaterals of mitral cells to the olfactory cortex was studied in the rabbit by intracellular staining with horseradish peroxidase (HRP). The stained mitral cell axons were reconstructed from the soma to the most caudal portion of the anterior piriform cortex (aPC). Single mitral cells projected to cytoarchitectonically different areas of the olfactory cortex, i.e., the anterior olfactory nucleus (AON), the aPC, and the olfactory tubercle (OT). All the stained mitrall cells projected to both the AON and the aPC, and about one-fourth of the mitral cells projected to the OT. At the surface of the AON and the aPC, the main axon running in the lateral olfactory tract (LOT) gave off several thin collaterals at various intervals. The collaterals did not project evenly in each area but typically formed patchy terminal arbors which tended to be elongated anteroposteriorly. In both the AON and the aPC, each single mitral cell formed several terminal arbors in layer Ia. The axon collaterals innervating the OT showed two types of projection patterns. One type of collateral was emitted from the main axon within the olfactory bulb, coursed through the ventro-medial portion of the olfactory peduncle without joining the main mass of the LOT and terminated mainly in the medial portion of the OT. The other type of collateral emerged from the main axon in the LOT, coursed medioposteriorly, and projected to the lateral portion of the OT. Although individual mitral cells projected to several parts of the olfactory cortex, the fact that they made dense terminal arbors in specific places in each area suggests that the bulbocortical connections are not diffuse but highly selective.

Distribution of local axon collaterals of mitral, displaced mitral, and tufted cells in the rabbit olfactory bulb.

To determine the projection fields of intrabulbar axon collaterals, mitral, displaced mitral, and middle tufted cells in the rabbit olfactory bulb were stained by intracellular injection of HRP. The axon collaterals of mitral cells and displaced mitral cells were distributed exclusively within the granule cell layer (GrL). Those of middle tufted cells were distributed mostly in the GrL and on rare occasions in the mitral cell layer. None of these collaterals entered the external plexiform layer. Axon collaterals of mitral cells, emitted at the depths of the GrL, were distributed widely from the deep portion to the most superficial portion of the GrL. Collaterals of displaced mitral cells were also emitted in the deep part, but they tended to be distributed more superficially in the GrL than mitral cells. Collaterals of middle tufted cells were released and distributed superficially in the GrL. The axon collaterals of these principal cells typically extended for a longer distance than the secondary dendrites, and they sometimes formed bushlike terminal arborizations. The results indicate that the projection fields of the axon collaterals of principal cells are spatially separated from the dendritic projection fields. This suggests that the output of these principal cells through the collaterals has a functionally different role from the output through the dendrites. The observation that the three types of principal cells differ in the distribution pattern of their axon collaterals in the GrL suggests that there is a functional separation of the sublayers in the GrL.

Distribution, morphology, and gamma-aminobutyric acid immunoreactivity of horizontally projecting neurons in the macaque inferior temporal cortex.

In area TE of the macaque inferior temporal cortex, horizontal axons running parallel to the pial surface mediate interactions between laterally displaced sites across the cortex. We examined the spatial distribution and the types of cells that give rise to these horizontal axons, which are important factors in determining the nature of the lateral interactions in TE. Intracortical injections of retrograde tracers labeled columnar clusters of cells and cells diffusely scattered within TE. The clusters were 0.35 +/- 0.11 mm (mean +/- SD) in diameter and were laterally distributed up to 6 mm from the injection site. Labeled cells were found in layers 2 to 6, with only a few labeled cells seen in layer 4. The clustering of labeled cells in layers 5 and 6 was looser than that in layers 2 and 3. Intracellular staining of the retrogradely labeled cells revealed that the majority of them were typical or modified pyramidal cells, both within and between the clusters. Only a few nonpyramidal interneurons were also stained at the fringe of the tracer injection site. Consistent with these results, only a small proportion of the retrogradely labeled cells exhibited gamma-aminobutyric acid (GABA)-like immunoreactivity, mostly found within 1 mm from the injection site. The results indicate that direct horizontal interactions in TE are predominantly mediated by pyramidal or modified pyramidal cells in layers 2, 3, 5, and 6 and are primarily excitatory in nature. The contribution of GABAergic interneurons to direct horizontal interactions is restricted to only short-distance projections.

Cortical convergence from different frequency domains in the cat primary auditory cortex.

Primary auditory cortex (AI) has a tonotopic map consisting of orderly isofrequency (IF) bands, and cortical connections are commonly supposed to link domains preferring similar characteristic frequencies (CFs) within AI and in auditory association cortex. The interaction of different frequency channels, however, has not fully been understood in terms of anatomical substrates. Here, by injecting two anterograde tracers in different frequency domains of cat AI, without overlap of the injection cores, we attempted to relate the anatomical mapping of cortical outputs to physiologically defined fields in the auditory cortex. Consistent with previous studies, patches of labeled axon terminals were oriented largely along the IF axis. In regions distant from the injection sites, however, terminal patches were divergent in distribution. This divergence resulted in a complex geometry of partial overlap of projections originating from the two injection sites. The relative extent of the overlap tended to vary depending on the distance between the two injection sites. Physiological mapping for tonotopy across auditory fields revealed that projectional overlap was characteristic of dorsal AI and the dorsoposterior field and, to a lesser extent, in the secondary auditory field. Considering the differences in frequency representation in different AI IF bands, the anatomical convergence of projections tuned to different CFs could contribute to the spectral integration of sound components. Furthermore, the different extent of convergence in the functionally distinct fields might reflect field-specific processing of acoustic signals.

Dendritic arborization patterns of cortical interneurons labeled with the lectin, Vicia villosa, and injected intracellularly with Lucifer yellow in aldehyde-fixed rat slices.

Neurons whose cell bodies had been stained by a lectin, Vicia villosa, which recognizes terminal N-acetylgalactosamine residue, were intracellularly injected with Lucifer yellow (LY), in aldehyde-fixed slices of the parietal cortex of rats. LY was subsequently visualized immunocytochemically. All injected neurons had smooth or only sparsely spiny dendrites and resembled the various forms of gamma-aminobutyric acid-ergic neurons previously described in rat neocortex. In layers II/III, IV and V, most injected neurons were multipolar. In layer VI, most had vertically elongated dendritic fields. Some injected neurons in layer IV had an oval or vertically elongated soma and a bitufted dendritic arborization pattern. There was a gradual increase in the overall dendritic extent in deeper layers of the cortex.

Two classes of cortical neurones labelled with Vicia villosa lectin in the guinea-pig.

Cell surface substance(s) containing terminal N-acetylgalactosamine (GaINAc) was localized in the cerebral cortex of the guinea-pig. Vicia villosa and Glycine max, both specific to GaINAc, labelled the surface of the cell body of multipolar and triangular neurones. The labelling extended to the apical dendrite and axon initial segment in the triangular neurones. They were distributed exclusively in layer 5. Most of the labelled multipolar neurones contained a calcium-binding protein, parvalbumin (Pv), while none of the triangular neurones did. The findings indicate that, in the guinea-pig, pyramidal neurones as well as non-pyramidal interneurones are enwrapped by GaINAc-containing substances and suggest that this enwrapping does not seem to be linked to Pv production.

Use of two anterograde axon tracers to label distinct cortical neuronal populations located in close proximity.

In order to investigate converging projections originating from adjacent populations of cortical neurons, injections of two different anterograde tracers, biotinylated dextranamine (BDA) and Phaseolus vulgaris leucoagglutinin (PHA-L), were made in close proximity. When the two injection sites were separated by around 500 microm and the time between injections was 1--4 h, BDA-labeling of neuronal elements was found not only at the BDA injection site but also at the PHA-L injection site. This false-positive BDA labeling of neurons at the PHA-L injection site was so intense that labeled axons could be traced, both into the neighboring cortical gray matter and into white matter. Increasing the separation distance to 1000 microm resulted in much fewer falsely positive labeled neurons at the PHA-L injection site. Even more effective was extending the time interval between the two injections. Thus, if the BDA injection preceded the PHA-L injection by more than 12 h, virtually no false-positive labeling was associated with the PHA-L injection site. These procedures may be applied to other combinations of anterograde tracers, such as BDA with tetramethylrhodamine-conjugated dextran amine.

Molecular heterogeneity of Vicia villosa-recognized perineuronal nets surrounding pyramidal and nonpyramidal neurons in the guinea pig cerebral cortex.

Of three monoclonal antibodies (mAbs Cat 301, 1B5 and 473) which recognize epitopes on perineuronal nets (PnNs), only mAb 473 displayed considerably varied degrees of staining intensity on Vicia villosa-labeled pyramidal (P) neurons (5%, intensely; 54%, very weakly; and 41%, unstained). These results indicate the heterogeneity on molecular structure or composition of the terminal N-acetylgalactosamine-containing PnNs within P class as well as between P and nonpyramidal classes.

Electron microscopic evidence that axon terminals from the mediodorsal thalamic nucleus make direct synaptic contacts with callosal cells in the prelimbic cortex of the rat.

A combined study of anterograde axonal degeneration and HRP retrograde labeling has shown that there exist monosynaptic connections between afferent fibers from the mediodorsal thalamic nucleus (MD) and callosal cells in the prelimbic cortex of the rat. Degenerating axon terminals from MD made asymmetrical synaptic contacts with dendritic spines from apical dendrites of layer III pyramidal cells that were retrogradely labeled with HRP after its injection into the prelimbic cortex contralateral to MD lesions.