Plant-produced idiotype vaccines for the treatment of non-Hodgkin's lymphoma: safety and immunogenicity in a phase I clinical study.

Plant-made vaccines have been the subject of intense interest because they can be produced economically in large scale without the use of animal-derived components. Plant-made therapeutic vaccines against challenging chronic diseases, such as cancer, have received little research attention, and no previous human clinical trials have been conducted in this vaccine category. We document the feasibility of using a plant viral expression system to produce personalized (patient-specific) recombinant idiotype vaccines against follicular B cell lymphoma and the results of administering these vaccines to lymphoma patients in a phase I safety and immunogenicity clinical trial. The system allowed rapid production and recovery of idiotypic single-chain antibodies (scFv) derived from each patient's tumor and immunization of patients with their own individual therapeutic antigen. Both low and high doses of vaccines, administered alone or co-administered with the adjuvant GM-CSF, were well tolerated with no serious adverse events. A majority (>70%) of the patients developed cellular or humoral immune responses, and 47% of the patients developed antigen-specific responses. Because 15 of 16 vaccines were glycosylated in plants, this study also shows that variation in patterns of antigen glycosylation do not impair the immunogenicity or affect the safety of the vaccines. Collectively, these findings support the conclusion that plant-produced idiotype vaccines are feasible to produce, safe to administer, and a viable option for idiotype-specific immune therapy in follicular lymphoma patients.

Substrate deformation in a hypoxanthine-guanine phosphoribosyltransferase ternary complex: the structural basis for catalysis.

BACKGROUND: Hypoxanthine-guanine phosphoribosyltransferases (HGPRTs) are well-recognized antiparasitic drug targets. HGPRT is also a paradigmatic representative of the phosphoribosyltransferase family of enzymes, which includes other important biosynthetic and salvage enzymes and drug targets. To better understand the reaction mechanism of this enzyme, we have crystallized HGPRT from the apicomplexan protozoan Toxoplasma gondii as a ternary complex with a substrate and a substrate analog. RESULTS: The crystal structure of T. gondii HGPRT with the substrate Mg2+-PRPP and a nonreactive substrate analog, 9-deazaguanine, bound in the active site has been determined at 1.05 A resolution and refined to a free R factor of 15.4%. This structure constitutes the first atomic-resolution structure of both a phosphoribosyltransferase and the central metabolic substrate PRPP. This pre-transition state complex provides a clearer understanding of the structural basis for catalysis by HGPRT. CONCLUSIONS: Three types of substrate deformation, chief among them an unexpected C2'-endo pucker adopted by the PRPP ribose ring, raise the energy of the ground state. A cation-pi interaction between Tyr-118 and the developing oxocarbenium ion in the ribose ring helps to stabilize the transition state. Enforced substrate propinquity coupled with optimal reactive geometry for both the substrates and the active site residues with which they interact contributes to catalysis as well.

Comparison of the actions of 9-beta-D-arabinofuranosyl-2-fluoroadenine and 9-beta-D-arabinofuranosyladenine on target enzymes from mouse tumor cells.

9-beta-D-Arabinofuranosyl-2-fluoroadenine (2-F-ara-A), a derivative of 9-beta-D-arabinofuranosyladenine (ara-A) that is resistant to deamination, selectively inhibits DNA synthesis and has activity against mouse leukemia L1210 comparable to that of ara-A plus the adenosine deaminase inhibitor, 2'-deoxycoformycin. To determine if these two nucleosides have similar modes of action, comparisons were made of their effects and those of their triphosphates on enzymes known to be inhibited by ara-A or 9-beta-D-arabinofuranosyladenine 5'-triphosphate. 9-beta-D-Arabinofuranosyl-2-fluoroadenine 5'-triphosphate was more effective than 9-beta-D-arabinofuranosyladenine 5'-triphosphate in inhibiting the reduction of adenosine 5'-diphosphate and cytidine 5'-diphosphate by ribonucleotide reductase from HEp-2 cells or L1210 cells. DNA polymerase alpha from L1210 cells was equally sensitive to 9-beta-D-arabinofuranosyl-2-fluoroadenine 5'-triphosphate and 9-beta-D-arabinofuranosyladenine 5'-triphosphate, and DNA polymerase beta from L1210 cells was much less sensitive to both triphosphates. S-Adenosylhomocysteine hydrolase from L1210 cells was inactivated by 2-F-ara-A and ara-A, but higher concentrations of the fluoro derivative were required. These results are consistent with 2-F-ara-A and ara-A inhibition of DNA synthesis by inhibition of ribonucleotide reductase and DNA polymerase alpha.

Effects of 2-chloro-9-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)adenine on K562 cellular metabolism and the inhibition of human ribonucleotide reductase and DNA polymerases by its 5'-triphosphate.

2-Chloro-9-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)-adenine (Cl-F-ara-A) has activity against the P388 tumor in mice on several different schedules. Biochemical studies with a chronic myelogenous leukemia cell line (K562) grown in cell culture have been done in order to better understand its mechanism of action. Cl-F-ara-A was a potent inhibitor of K562 cell growth. Only 5 nM inhibited K562 cell growth by 50% after 72 h of continuous incubation. The 5'-triphosphate of Cl-F-ara-A was detected by strong anion exchange chromatography of the acid-soluble extract of K562 cells incubated with Cl-F-ara-A. Competition studies with natural nucleosides suggested that deoxycytidine kinase was the enzyme responsible for the metabolism to the monophosphate. Incubation of K562 cells for 4 h with 50 nM Cl-F-ara-A inhibited the incorporation of [3H]thymidine into the DNA by 50%. Incubation with 0.1, 1, or 10 microM Cl-F-ara-A for 4 h depressed dATP, dCTP, and dGTP pools but did not affect TTP pools. Similar inhibition of deoxyribonucleoside triphosphate pools was seen after incubation with 2-chloro-2'-deoxyadenosine. Both Cl-F-ara-ATP and Cl-dATP potently inhibited the reduction of ADP to dADP in crude extracts of K562 cells (concentration producing 50% inhibition, 65 nM). The effect of Cl-F-ara-ATP on human DNA polymerases alpha, beta, and gamma isolated from K562 cells grown in culture was determined and compared with those of Cl-dATP and 9-beta-D-arabinofuranosyl-2-fluoroadenine triphosphate (F-ara-ATP). Cl-F-ara-ATP was a potent inhibitor of DNA polymerase alpha. Inhibition of DNA polymerase alpha was competitive with respect to dATP (Ki of 1 microM). The three analogue triphosphates were incorporated into the DNA by DNA polymerase alpha as efficiently as dATP. The incorporation of Cl-F-ara-AMP inhibited the further elongation of the DNA chain, similarly to that seen after the incorporation of F-ara-AMP. Extension of the DNA chain after the incorporation of Cl-dAMP was not inhibited as much as it was with either Cl-F-ara-AMP or F-ara-AMP. Cl-F-ara-ATP was not a potent inhibitor of DNA polymerase beta, DNA polymerase gamma, or DNA primase.(ABSTRACT TRUNCATED AT 400 WORDS)

Specific methods to identify plasma binding abnormalities in euthyroid hyperthyroxinemia.

Methods to identify the plasma T4-binding abnormalities that can cause euthyroid hyperthyroxinemia were evaluated in patients with excess T4-binding globulin, familial dysalbuminemic hyperthyroxinemia, prealbumin-associated hyperthyroxinemia, and autoantibody binding of T4. Familial dysalbuminemic hyperthyroxinemic serum showed a unique persistence of abnormal [125I]T4 binding when diluted 1:100 in phosphate buffer with added 1000-fold excess of unlabeled T4 (10(-6) M T4). Immunoprecipitation of [125I]T4 by antibody to prealbumin, precipitation of [125I]T4 by polyethylene glycol 6000 19%, and in vitro resin uptake of T3 were specific for prealbumin-associated hyperthyroxinemia, autoantibody binding of T4, and T4-binding globulin excess, respectively. These simple methods facilitate investigation of patients with euthyroid hyperthyroxinemia and will identify individuals and families at risk of misdiagnosis by standard methods. Use of these techniques rules out the known binding abnormalities in hyperthyroxinemic patients and may make the diagnosis of generalized hormone resistance more specific.

Familial euthyroid thyroxine excess: characterization of abnormal intermediate affinity thyroxine binding to albumin.

The abnormal high capacity T4 binding site of familial euthyroid T4 excess was separable from prealbumin and T4-binding globulin but not from albumin. We therefore compared T4 binding by albumin preparations isolated from the sera of normal and affected subjects. By equilibrium dialysis, albumin from affected subjects showed an extra T4 binding site (Kd approximately 50 nM) in addition to the T4 binding sites of normal albumin (Kd approximately 4 microM). Comparison of the estimated capacity of the additional site (200 microM) with the molar concentration of albumin suggested that only about one third of albumin molecules from affected subjects contained the extra binding site. Estimates of affinity and capacity were used to derive combining powers for the diverse classes of serum T4 binding sites. From these estimates, it appears that the presence of the abnormal site accounts for the approximate doubling of normal mean total T4 (from approximately 100 nM or 7.7 micrograms/dl to approximately 200 nM or 15.5 micrograms/dl), in order to maintain a normal free T4 in the face of the increased T4 association with albumin. Studies of [125I]T4 displacement from albumin of affected subjects showed low T3 affinity and competition by barbitone. Relative molar concentrations to give equivalent displacement of [125I]T4 were: 3,3',5,5'-tetraiodothyroacetic acid, 0.4; T4, 1.0; rT3, 4; 8-anilinonaphthalene sulfonic acid, 10; T3, 80; salicylate, 200; and barbitone, 40,000. Studies with dithiothreitol suggested that disulfide bonds were critical in maintaining the T4-albumin association. These findings indicate that familial T4 excess is due to abnormal intermediate affinity, sulfhydryl-sensitive T4 binding sites that are inseparable from the albumin found in affected subjects.

Evaluation of free thyroxine methods in the presence of iodothyronine-binding autoantibodies.

Circulating iodothyronine-binding autoantibodies interfere with total T4 and T3 RIAs, giving falsely high or falsely low values depending on the assay separation method used. Direct serum free T4 (FT4) measurement should compensate for such abnormal binding if the method is free of artefact. We assessed 5 different FT4 methods in 12 patients with immunoglobulin binding of iodothyronines, with limited assessment of a sixth method in 2 subjects. Thyroid status was assessed clinically and by measurement of TSH and its response to TRH. T4 methods which use analog tracers, i.e. Amerlex, and Clinical Assays One-step FT4, gave spuriously high values in almost all hypothyroid and some euthyroid patients, due to immunoglobulin binding of the tracers used in these techniques. The kinetic indirect method (Corning Immunophase FT4) gave inappropriately high values in 4 of 6 hypothyroid patients. FT4 by equilibrium dialysis or by adsorption chromatography and RIA, accurately assessed thyroid status. These findings suggest that FT4 methods are valid in patients with circulating iodothyronine-binding immunoglobulins only if the free hormone fraction is physically separated from serum binding proteins before the assay procedure.

A pituitary tumor producing high molecular weight adrenocorticotropin-related peptides: clinical and cell culture studies.

The tissue-specific processing of proopiomelanocortin (POMC), the precursor of ACTH, beta-endorphin, and their related peptides, is currently of considerable interest. We report a patient with a large aggressive pituitary tumor, Cushing's syndrome, and hyperpigmentation managed by transsphenoidal hypophysectomy, bilateral adrenalectomy, and sellar irradiation. Preoperatively, plasma levels of immunoreactive ACTH (ir-ACTH; 280 ng/liter) and beta-endorphin (ir-beta EP; 520 ng/liter) were moderately elevated. Chromatography of the plasma showed two peaks of ACTH immunoreactivity, with the major peak eluting in the void volume, and two major peaks of ir-beta EP, corresponding to the elution positions of beta-lipotropin and beta-endorphin standards. Plasma ir-ACTH and ir-beta EP were not suppressed by high doses of glucocorticoid or bromocriptine, a degree of autonomy more commonly found with POMC production from ectopic sources than that from pituitary tumors. Tissue removed at operation was enzymatically dispersed, and the cells were cultured in suspension, propagated, and passaged sequentially for over 20 passages. Using this cell line, we demonstrated that the biosynthesis of POMC, its pattern of processing, and the release of POMC/ir-beta EP/ir-ACTH in vitro were consistent with the in vivo evidence of autonomous secretion and abnormal processing of POMC by this pituitary tumor.

Familial euthyroid thyroxine excess: an appropriate response to abnormal thyroxine binding associated with albumin.

Responses of the pituitary-thyroid axis and T4 binding to plasma proteins were studied in three kindreds with familial euthyroid T4 excess, an autosomal dominant condition in which affected subjects have high concentrations of plasma T4 with a high free T4 index, but normal free T4 by equilibrium dialysis. Treatment of affected subjects with exogenous T4 or T3 led to gradual suppression of TSH secretion when the free level of T4 or T3 increased above normal. When total T4 was reduced toward normal by potassium iodide treatment or previous subtotal thyroidectomy, the findings suggested mild hormone deficiency. In affected subjects from all three families, equilibrium dialysis showed increased [125I]T4 binding, with evidence of abnormal high capacity binding when an excess of unlabeled T4 was added. In contrast, T3 binding showed no major abnormality. Serum concentrations of T4-binding globulin, prealbumin, and albumin were normal, but gel electrophoresis and immunoprecipitation of binding proteins indicated that 25-30% of tracer [125I]T4 was albumin bound (normal, 10-12%). Abnormal binding, studied by an adsorption separation system in the presence of T4 excess, was inhibited by increments of barbitone. These findings suggest that T4 excess is an appropriate response to abnormal T4 binding so as to maintain normal free T4. The excess bound T4 is associated with a normal quantity of albumin. The basis for increased T4-albumin binding remains to be determined.

Synaptic connections of callosal projection neurons in the vibrissal region of mouse primary motor cortex: an electron microscopic/horseradish peroxidase study.

Reciprocal axonal projections between homotypic areas of the vibrissal region of mouse primary motor cortex (MsI) (Porter and White: Neurosci. Lett. 47:37-40, '84) suggested the existence of reciprocal synaptic connections between callosal projection neurons and callosal afferents. In the present study, the retrograde transport of horseradish peroxidase (HRP) was combined with lesion-induced degeneration to identify synapses between callosal afferents and callosal neurons in the corresponding region of the contralateral cortex. The procedure was as follows: MsI was injected with HRP and aspirated on the following day. After 4 days, the animals were perfused and motor cortex was processed for HRP according to a variation of the Adams (Brain Res. 176:33-47,'77) technique, and postfixed in OsO4. The methods used consistently filled fine dendritic branches and spines with dense reaction product, thus allowing examination of synaptic contacts with these processes. All callosal projection neurons were identified as pyramidal neurons, having somata in cortical layers II/III and V. Labeled cells from each of the two levels were prepared for electron microscopy, and that part of each cell's apical dendrite that traversed the superficial cortical layers, where most callosal axons terminate, was cut in an unbroken series of thin sections. Micrographs were taken of all labeled profiles in each thin section, and tracings of the profiles were assembled to reconstruct the apical dendrites. Data on the distribution, type, and amount of callosal and other synapses with the shaft and spines of the apical dendrites were obtained by examining the reconstructions. In addition, profiles of basal dendrites of layer II/III cells were examined in thin sections to ascertain the numbers of callosal and other synapses formed with their shafts and spines. The proportion of synapses that each dendrite formed with callosal axon terminals was compared to the concentration of callosal afferents in the neuropil. Dendrites of both layer II/III and layer V pyramidal cells synapsed with callosal axon terminals. The apical and basal dendrites of layer II/III neurons formed a similar proportion of their synapses with callosal afferents, and this was similar to the concentration of callosal synapses in the surrounding neuropil. Segments of apical dendrites belonging to layer V and layer II/III neurons course through neuropil containing nearly the same concentration of degenerating callosal terminals, but the layer V cells form fewer callosal synapses.(ABSTRACT TRUNCATED AT 400 WORDS)

Afferent and efferent pathways of the vibrissal region of primary motor cortex in the mouse.

The afferent and efferent connections of the vibrissal representation within the mouse primary motor cortex (MsI) were identified by using the retrograde transport of horseradish peroxidase (HRP) and the anterograde transport of tritiated amino acids injected into MsI. Following aldehyde perfusion brains were frozen-sectioned at 40 microns and reacted for HRP using the 3-3' diaminobenzidine-cobalt chloride technique of Adams ('77). Alternate HRP reacted sections were processed for autoradiography. HRP-filled pyramidal cell somata and concentrations of developed silver grains above background levels were observed in both the vibrissal area of primary somatosensory cortex (SmI) cortex (i.e., the posteromedial barrel subfield; PMBSF cortex) and in the face region of SmII (area 40). In both regions labeled somata occurred predominantly in cortical layers II-III and V. Autoradiographic label was superimposed over the regions containing labeled somata but exhibited a less distinct laminar organization. A dense reciprocal projection connected the injection site with the homotopic area in contralateral MsI; somata occurred for the most part in layers III and V. Developed silver grains were uniformly dispersed over the area containing labeled cell bodies. HRP-labeled pyramidal somata were noted in contralateral PMBSF cortex, but no silver grains occurred in this region. Reciprocal projections linked MsI cortex with the ipsilateral thalamic nuclei: ventralis pars lateralis (VL) and centralis pars lateralis (CL) and with the zona incerta (ZI). Labeled cell bodies and developed silver grains were more dense in VL than in CL. The ipsilateral striatum and thalamic reticular nucleus (NRT) received afferents from the motor cortex but did not project to it. Thus, the vibrissal area of primary motor cortex is connected with a number of cortical and subcortical structures, each of which has been shown to play a role in motor performance. Identification of the afferent and efferent pathways of MsI cortex will now enable further investigation of the ultrastructural and synaptic organization of the vibrissal area of MsI.

Intrinsic circuitry involving the local axon collaterals of corticothalamic projection cells in mouse SmI cortex.

The objective of this study was to identify the components involved in a local synaptic circuit in the mouse cerebral cortex. The local axon collaterals of corticothalamic (CT) projection cells in the posteromedial barrel subfield area of primary somatosensory cortex were labeled by the retrograde transport of horseradish peroxidase injected into the ipsilateral thalamus. Thalamocortical (TC) axon terminals in the same region of cortex were labeled by lesion induced degeneration. CT axon terminals synapsed preferentially with dendritic shafts, whereas TC axon terminals synapsed mainly with dendritic spines. Some dendrites received both CT and TC synapses. Dendrites were interpreted to belong to nonspiny multipolar cells. These results indicate that a reciprocal synaptic relationship exists in the cortex between nonspiny multipolar cells and CT projection cells. Both CT projection cells and nonspiny multipolar neurons have been shown previously to receive TC synapses (White and Hersh: J. Neurocytol. 11:137-157, '82; White, Benshalom, and Hersch: J. Comp. Neurol. 229:311-320, '84). These findings imply that a triadic relationship involving afferent input and populations of CT projection and intrinsic neurons is a basic feature of the synaptic organization of the cerebral cortex.

Comparison of the distribution of parvalbumin-immunoreactive and other synapses onto the somata of callosal projection neurons in mouse visual and somatosensory cortex.

The distribution of synapses made by parvalbumin-immunoreactive (pv-ir) and nonimmunoreactive terminals was determined for the cell bodies of callosal projection neurons in the somatosensory and visual areas of mouse cerebral cortex. Callosal neurons were labeled by the retrograde transport of horseradish peroxidase applied to the contralateral hemisphere. The surface areas of somata belonging to callosal cells in somatosensory cortex ranged from 230 to 243 microm2 in size and received roughly one-third of their synapses from pv-ir terminals. Visual cortex, in contrast, contained two populations of callosal cell bodies: relatively large ones ranging in size from 255 to 279 microm2 that received 3-9% of their synapses from pv-ir terminals and smaller cell bodies that both in size (232-237 microm2) and in the proportion of synapses received from pv-ir terminals resemble the callosal cells examined in somatosensory cortex. That different functional areas of the cortex have populations of callosal cells similar in size, and displaying similar patterns of somatic synapses, supports the notion that a common plan of synaptic connectivity characterizes different functional areas. Results in visual cortex indicate that functional areas contain, in addition, area-specific patterns of synapses.

Synapses made by axons of callosal projection neurons in mouse somatosensory cortex: emphasis on intrinsic connections.

This is one of a series of papers aimed at identifying the synaptic output patterns of the local and distant projections of subgroups of pyramidal neurons. The subgroups are defined by the target site to which their main axon projects. Pyramidal neurons in areas 1 and 40 of mouse cerebral cortex were labeled by the retrograde transport of horseradish peroxidase (HRP) transported from severed callosal axons in the contralateral hemisphere. Terminals of the local axon collaterals of these neurons ("intrinsic" terminals) were identified in somatosensory areas 1 and 40, and their distribution and synaptic connectivity were examined. Also examined were the synaptic connections of "extrinsic" callosal axon terminals labeled by lesion induced degeneration consequent to the severing of callosal fibers. A post-lesion survival time of 3 days was chosen because by this time the extrinsic terminals were all degenerating, whereas the intrinsic terminals were labeled by HRP. Both intrinsic and extrinsic callosal axon terminals occurred in all layers of the cortex where they formed only asymmetrical synapses. Layers II and III contained the highest concentrations of both types of callosal axon terminal. Analyses of serial thin sections through layers II and III in both areas 1 and 40 yielded similar results: 97% of the extrinsic (277 total sample) and of the intrinsic (1215 total sample) callosal axon terminals synapsed onto dendritic spines, likely those of pyramidal neurons; the remainder synapsed onto dendritic shafts of both spiny and nonspiny neurons. Thus the synaptic output patterns of intrinsic vs. extrinsic callosal axon terminals are strikingly similar. Moreover, the high proportion of axospinous synapses formed by both types of terminal contrasts with the proportion of asymmetrical, axospinous synapses that occur in the surrounding neuropil where only about 80% of the asymmetrical synapses are onto spines. This result is in accord with previous quantitative studies of the synaptic connectivities of both extrinsic and intrinsic axonal pathways in the cortex (White and Keller, 1989: Cortical Circuits; Boston: Birkhauser): in all instances, axonal pathways are highly selective for the types of elements with which they synapse.

Intrinsic circuitry: synapses involving the local axon collaterals of corticocortical projection neurons in the mouse primary somatosensory cortex.

Pyramidal neurons in the mouse SmI cortex were labeled by the retrograde transport of horseradish peroxidase (HRP) injected into the ipsilateral MsI cortex. Terminals of the local axon collaterals of these neurons (CC terminals) were identified in SmI, and their distribution and synaptic connectivity were examined. To avoid confusion, terminals in SmI cortex labeled by the anterograde transport of HRP injected into MsI were eliminated by lesion-induced degeneration. Lesions of MsI were made 24 hours after the injection of HRP; postlesion survival time was 4 days. Most CC axon terminals occurred in layers III and V where they formed asymmetrical synapses. Of 139 CC synapses in layer III and 104 in layer V, approximately 13% were formed with dendritic shafts. Reconstruction of 19 of these dendrites from serial thin sections showed them to originate from both spiny and nonspiny neurons. Most synapses of CC terminals (about 87%) were onto dendritic spines. In contrast, White and Keller (1987) demonstrated that terminals belonging to the local axon collaterals of corticothalamic (CT) projection cells synapse mainly with dendritic shafts of nonspiny neurons: 92% onto shafts, the remainder onto spines. The distribution of asymmetical synapses onto spines and dendritic shafts was analyzed for neuropil in layers III, IV, and V. Depending on the layer, from 34 to 46% of the asymmetrical synapses in the neuropil were onto dendritic shafts. Results showing that CC and CT terminals form proportions of axodendritic vs. axospinous synapses that differ from each other, and from the neuropil, indicate that local axon collaterals are highly selective with regard to their postsynaptic elements.

Synapses of extrinsic and intrinsic origin made by callosal projection neurons in mouse visual cortex.

Neurons in areas 17/18a and 17/18b of mouse cerebral cortex were labeled by the retrograde transport of horseradish peroxidase (HRP) transported from severed callosal axons in the contralateral hemisphere. Terminals of the local axon collaterals of labeled neurons (intrinsic terminals) were identified in the border regions of area 17 with areas 18a and 18b, and their distribution and synaptic connectivity were determined. Also examined were the synaptic connections of extrinsic callosal axon terminals labeled by lesion-induced degeneration consequent to the severing of callosal fibers. A postlesion survival time of 3 days was chosen because by this time the extrinsic terminals were all degenerating, whereas the intrinsic terminals were labeled by horseradish peroxidase. Both intrinsic and extrinsic callosal axon terminals occurred in all layers of the cortex where, with rare exception, they formed asymmetrical synapses. Layers II and III contained the highest concentrations of intrinsic and extrinsic callosal axon terminals. Analyses of serial thin sections through layers II and III in both areas 17/18a and 17/18b yielded similar results: 97% of the intrinsic (1,412 total sample) and of the extrinsic (414 total sample) callosal axon terminals synapsed onto dendritic spines, likely those of pyramidal neurons; the remainder synapsed onto dendritic shafts of both spiny and nonspiny neurons. Thus, the synaptic output patterns of intrinsic vs. extrinsic callosal axon terminals are strikingly similar. Moreover, the high proportion of axospinous synapses formed by both types of terminal (97%) contrasts with the proportion of asymmetrical axospinous synapses that occurs in the surrounding neuropil where about 64% of the asymmetrical synapses are onto spines. This result is in accord with previous quantitative studies of the synaptic connectivities of callosal projection neurons in mouse somatosensory cortex, and lends additional weight to the hypothesis that axonal pathways are highly selective for the types of elements with which they synapse.

Cortical modules in the posteromedial barrel subfield (Sml) of the mouse.

An antibody to MAP2 was used on sections through the posteromedial barrel subfield (PMBSF) of primary somatosensory cortex to reveal the distributions of cell bodies and dendrites. It was found that apical dendrites of layer VI neurons form irregular bundles or sheets that break up in layer IV, where most of these dendrites form their terminal tufts. In contrast, the apical dendrites of layer V pyramidal neurons form clusters that ascend into layer II/III where they are joined by apical dendrites of the superficial pyramidal neurons. In layer IV the clusters of the layer V apical dendrites are more concentrated in barrel walls than in hollows. Thus, in layer IV the average center to center spacing between the clusters is about 25 microns in the barrel hollows, and about 22 microns in the barrel walls. In part, this differential distribution of the apical dendritic clusters is brought about because the apical dendrites of layer V pyramids beneath the periphery of the barrel hollows arc towards the barrel walls as they pass from layer V into layer IV. Based on previous analyses of the three-dimensional organization of the primary visual areas in the monkey, cat, and rat, it has been proposed that neurons in these cortices are organized into modules that are centered on the clusters of apical dendrites belonging to layer V pyramidal neurons. Mouse PMBSF cortex is composed of similar pyramidal cell modules and the organization of neurons in these modules is similar to that in visual cortex. This suggests that the pyramidal cell modules are fundamental neuronal units that exist throughout the cerebral cortex, and implies that the various functional areas of the cortex in different species are organized according to a common, basic plan.

Thalamocortical synapses involving identified neurons in mouse primary somatosensory cortex: a terminal degeneration and golgi/EM study.

Synapses involving thalamocortical afferents and hitherto unexamined neuron types of the posteromedial barrel subfield (PMBSF) of the mouse have been identified using a combined degeneration and Golgi/EM technique (Peters et al., '77). Degeneration of thalamocortical axon terminals was produced with electrolytic lesions of the nucleus ventralis posterior, pars lateralis thalami, and the nucleus posterior thalami. Four days after receiving lesions, the animals were perfused, and blocks of cortex containing the PMBSF were processed by the Golgi method. The blocks were tissue-chopped at 125 microns and examined with the light microscope. Sections containing neurons of interest were gold-toned and deimpregnated in preparation for electron microscopy (Fairén et al., '77). Portions of selected neurons contained in layers III-IV were serially thin-sectioned and examined with an electron microscope to determine if they were involved in synapses with degenerating thalamocortical axon terminals. Results showed thalamocortical synapses on the apical dendrites of five different sized pyramidal cells whose somata occurred in layers V and VI, and on dendrites of on spiny bitufted neuron and one non-spiny multipolar neuron with somata in layer V. A non-spiny bitufted neuron of layer IV which was not impregnated also received thalamocortical synapses. Although every neuron examined formed at least one thalamocortical synapse, some formed very few, whereas others formed many. Of the pyramidal cells, small layer V and VI pyramidal cells and a large deep layer V pyramidal cell were involved in small numbers of thalamocortical synapses, while a medium superficial layer V pyramidal cell and a large layer VI pyramidal cell each formed many. The spiny bitufted neuron formed a small number of thalamocortical synapses. The findings suggest that, whereas any type of neuron with a dendrite in layer IV likely receives some synaptic input from the thalamus, individual neurons were involved in very different quantities of thalamocortical synapses.

Thalamocortical synapses of pyramidal cells which project from SmI to MsI cortex in the mouse.

Electrolytic lesions were made of the nucleus ventralis posterior pars lateralis thalami and of the nucleus posterior thalami in the male CD/1 mice to label thalamocortical axon terminals in mouse SmI cortex. Pyramidal cells in layers II through V of SmI cortex were labeled by the retrograde transport of horseradish peroxidase injected into ipsilateral MsI. Numerous pyramidal cells, particularly in the more superficial layers of SmI cortex, were filled with reaction product so that even their dendritic spines and local axon collaterals were clearly visible. Six well-filled pyramidal cells with somata in layers III and IV were serial thin-sectioned, and portions of their dendrites in layer IV were examined with the electron microscope to determine the distribution of their thalamocortical and other synapses. It was found that, in general, different dendrites of a single pyramidal cell formed similar proportions of thalamocortical synapses and that the six pyramidal cells, as a group, also formed similar proportions of thalamocortical synapses with their dendrites in layer IV. In contrast, when the thalamocortical connectivity of the six cells was considered as a function of their depths within the cortex, a clear trend was seen for the proportion of thalamocortical synapses to increase with increasing proximity of the cell body to layer IV. A hypothesis based on the timing of developmental events is proposed to account for this observation.

A comparison of thalamocortical and other synaptic inputs to dendrites of two non-spiny neurons in a single barrel of mouse SmI cortex.

A Golgi impregnated, non-spiny multipolar cell whose soma occurred in layer V of the region of mouse SmI cortex containing the posteromedial barrel subfield (PMBSF) (Woolsey and Van der Loos, '70) was gold-toned and deimpregnated (Fairen et al., '77). Two of its dendrites, contained within a single PMBSF barrel, were serial thin-sectioned and then reconstructed in three dimensions. Dendrites of an unimpregnated, non-spiny layer IV bitufted cell, present within the same barrel, were also reconstructed in three dimensions from the series of thin sections. This approach permitted a comparison of the distribution of synapses along dendrites of the two non-spiny neurons. Results showed dendrites of the layer IV bitufted cell formed about twice as many synapses per unit length as those of the multipolar cell. Particularly striking was the contrast between the large number of synapses made by degenerating thalamocortical axon terminals with the dendrites of the bitufted cell and the rarity with which such synapses occur on dendrites of the multipolar cell. Furthermore, the proportion of the total number of synapses made by thalamocortical axons terminals onto dendrites of the bitufted cell was six times greater than the proportion of the thalamocortical synapses onto the multipolar cell dendrites.