Specialization in pyramidal cell structure in the cingulate cortex of the Chacma baboon (Papio ursinus): An intracellular injection study of the posterior and anterior cingulate gyrus with comparative notes on the macaque and vervet monkeys

This study forms part of an ongoing investigation of pyramidal cell structure in the cingulate cortex of primates. Recently we have demonstrated that layer III pyramidal cells in the anterior cingulate gyrus are considerably larger, more branched and more spinous than those in the posterior cingulate gyrus (areas 24 and 23, respectively) in the macaque and vervet monkeys. Moreover, the extent of the interareal difference in specialization in pyramidal cell structure differed between the two species. These data suggest that pyramidal cell circuitry may have evolved differently in these closely related species. Presently there are too few data to speculate on what is selecting for this specialization in structure. Here we extend the basis for comparison by studying pyramidal cell structure in cingulate gyrus of the Chacma baboon (Papio ursinus). Methodology used here is the same as that for our previous studies: intracellular injection of Lucifer Yellow in flat-mounted cortical slices. We found that pyramidal cells in anterior cingulate gyrus (area 24) were more branched and more spinous than those in posterior cingulate gyrus (area 23). Moreover, the complexity in pyramidal cell structure in both the anterior and posterior cingulate gyrus of the baboon differed to that in the corresponding regions in either the macaque or vervet monkeys. (C) 2005 Elsevier Ireland Ltd. All rights reserved.

Regional specialization in pyramidal cell structure in the visual cortex of the galago: An intracellular injection study of striate and extrastriate areas with comparative notes on New World and Old World monkeys

Recent studies have revealed marked differences in the basal dendritic structure of layer III pyramidal cells in the cerebral cortex of adult simian primates. In particular, there is a consistent trend for pyramidal cells of increasing complexity with anterior progression through occipitotemporal cortical visual areas. These differences in pyramidal cell structure, and their systematic nature, are believed to be important for specialized aspects of visual processing within, and between, cortical areas. However, it remains unknown whether this regional specialization in the pyramidal cell phenotype is unique to simians, is unique to primates in general or is widespread amongst mammalian species. In the present study we investigated pyramidal cell structure in the prosimian galago (Otolemur garnetti). We found, as in simians, that the basal dendritic arbors of pyramidal cells differed between cortical areas. More specifically, pyramidal cells became progressively more spinous through the primary (V1), second (V2), dorsolateral (DL) and inferotemporal ( IT) visual areas. Moreover, pyramidal neurons in V1 of the galago are remarkably similar to those in other primate species, in spite of large differences in the sizes of this area. In contrast, pyramidal cells in inferotemporal cortex are quite variable among primate species. These data suggest that regional specialization in pyramidal cell phenotype was a likely feature of cortex in a common ancestor of simian and prosimian primates, but the degree of specialization varies between species. Copyright (C) 2005 S. Karger AG, Basel.

Regional specialization in pyramidal cell structure in the limbic cortex of the vervet monkey (Cercopithecus pygerythrus): an intracellular injection study of the anterior and posterior cingulate gyrus

The pyramidal cell phenotype varies quite dramatically in structure among different cortical areas in the primate brain. Comparative studies in visual cortex, in particular, but also in sensorimotor and prefrontal cortex, reveal systematic trends for pyramidal cell specialization in functionally related cortical areas. Moreover, there are systematic differences in the extent of these trends between different primate species. Recently we demonstrated differences in pyramidal cell structure in the cingulate cortex of the macaque monkey; however, in the absence of other comparative data it remains unknown as to whether the neuronal phenotype differs in cingulate cortex between species. Here we extend the basis for comparison by studying the structure of the basal dendritic trees of layer III pyramidal cells in the posterior and anterior cingulate gyrus of the vervet monkey (Brodmann's areas 23 and 24, respectively). Cells were injected with Lucifer Yellow in flat-mounted cortical slices, and processed for a light-stable DAB reaction product. Size, branching pattern, and spine density of basal dendritic arbors were determined, and somal areas measured. As in the macaque monkey, we found that pyramidal cells in anterior cingulate gyrus (area 24) were more branched and more spinous than those in posterior cingulate gyrus (area 23). In addition, the extent of the difference in pyramidal cell structure between these two cortical regions was less in the vervet monkey than in the macaque monkey.

Transient expression of a novel serine protease in the ectoderm of the ascidian Herdmania momus during development

We have studied gene expression during ascidian embryonic development using the technique of differential display and isolated partial cDNA sequences of 12 genes. Developmental regulation of these genes has been confirmed by northern hybridization analysis. Further cDNA cloning and sequence analysis of an mRNA that is present during gastrulation, neurulation and tailbud formation reveals that it encodes a novel serine protease containing a single kringle motif and catalytic domain. The spatial expression of this gene, designated Hmserp1, is restricted to precursor cells of the epidermis. The structure and expression of Hmsery1 is discussed in relation to possible functions during development.

Interlaminar differences in the pyramidal cell phenotype in cortical areas 7m and STP (the superior temporal polysensory area) of the macaque monkey

Pyramidal neurones were injected with Lucifer Yellow in slices cut tangential to the surface of area 7m and the superior temporal polysensory area (STP) of the macaque monkey. Comparison of the basal dendritic arbors of supra- and infragranular pyramidal neurones (n=139) that were injected in the same putative modules in the different cortical areas revealed variation in their structure. Moreover, there were relative differences in dendritic morphology of supra- and infragranular pyramidal neurones in the two cortical areas. Shell analyses revealed that layer III pyramidal neurones in area STP had considerably higher peak complexity (maximum number of dendritic intersections per Shell circle) than those in layer V, whereas peak complexities were similar for supra- and infragranular pyramidal neurones in area 7m. In both cortical areas, the basal dendritic trees of layer m pyramidal neurones were characterized by a higher spine density than those in layer V. Calculations of the total number of dendritic spines in the average basal dendritic arbor revealed that layer V pyramidal neurones in area 7m had twice as many spines as cells in layer III. (4535 and 2294, respectively). A similar calculation for neurones in area STP revealed that layer III pyramidal neurones had approximately the same number of spines as cells in layer V (3585 and 3850 spines, respectively). Relative differences in the branching patterns of, and the number of spines in, the basal dendritic arbors of supra- and infragranular pyramidal neurones in the different cortical areas may allow for integration of different numbers of inputs, and different degrees of dendritic processing. These results support the thesis that intra-areal circuitry differs in different cortical areas.

Protease modulation of the activity of the epithelial sodium channel expressed in Xenopus oocytes.

We have investigated the effect of extracellular proteases on the amiloride-sensitive Na+ current (INa) in Xenopus oocytes expressing the three subunits alpha, beta, and gamma of the rat or Xenopus epithelial Na+ channel (ENaC). Low concentrations of trypsin (2 microg/ml) induced a large increase of INa within a few minutes, an effect that was fully prevented by soybean trypsin inhibitor, but not by amiloride. A similar effect was observed with chymotrypsin, but not with kallikrein. The trypsin-induced increase of INa was observed with Xenopus and rat ENaC, and was very large (approximately 20-fold) with the channel obtained by coexpression of the alpha subunit of Xenopus ENaC with the beta and gamma subunits of rat ENaC. The effect of trypsin was selective for ENaC, as shown by the absence of effect on the current due to expression of the K+ channel ROMK2. The effect of trypsin was not prevented by intracellular injection of EGTA nor by pretreatment with GTP-gammaS, suggesting that this effect was not mediated by G proteins. Measurement of the channel protein expression at the oocyte surface by antibody binding to a FLAG epitope showed that the effect of trypsin was not accompanied by an increase in the channel protein density, indicating that proteolysis modified the activity of the channel present at the oocyte surface rather than the cell surface expression. At the single channel level, in the cell-attached mode, more active channels were observed in the patch when trypsin was present in the pipette, while no change in channel activity could be detected when trypsin was added to the bath solution around the patch pipette. We conclude that extracellular proteases are able to increase the open probability of the epithelial sodium channel by an effect that does not occur through activation of a G protein-coupled receptor, but rather through proteolysis of a protein that is either a constitutive part of the channel itself or closely associated with it.

Dendritic thickness: a morphometric parameter to classify mouse retinal ganglion cells

To study the dendritic morphology of retinal ganglion cells in wild-type mice we intracellularly injected these cells with Lucifer yellow in an in vitro preparation of the retina. Subsequently, quantified values of dendritic thickness, number of branching points and level of stratification of 73 Lucifer yellow-filled ganglion cells were analyzed by statistical methods, resulting in a classification into 9 groups. The variables dendritic thickness, number of branching points per cell and level of stratification were independent of each other. Number of branching points and level of stratification were independent of eccentricity, whereas dendritic thickness was positively dependent (r = 0.37) on it. The frequency distribution of dendritic thickness tended to be multimodal, indicating the presence of at least two cell populations composed of neurons with dendritic diameters either smaller or larger than 1.8 µm ("thin" or "thick" dendrites, respectively). Three cells (4.5%) were bistratified, having thick dendrites, and the others (95.5%) were monostratified. Using k-means cluster analysis, monostratified cells with either thin or thick dendrites were further subdivided according to level of stratification and number of branching points: cells with thin dendrites were divided into 2 groups with outer stratification (0-40%) and 2 groups with inner (50-100%) stratification, whereas cells with thick dendrites were divided into one group with outer and 3 groups with inner stratification. We postulate, that one group of cells with thin dendrites resembles cat ß-cells, whereas one group of cells with thick dendrites includes cells that resemble cat a-cells.

Aspectos morfológicos comparativos entre neurônios da camada I do córtex visual de duas espécies de roedores: Cavia porcellus e Rattus norvegicus

A camada I tem como característica principal a baixa quantidade de neurônios e uma alta densidade de fibras nervosas. A morfologia dos neurônios da camada I ainda é pouco estudada, tanto que nos estudos que avaliaram a morfologia desses neurônios não se chegou ainda a um consenso sobre as formas e funções desses neurônios. Este estudo avaliou a morfologia dos neurônios da camada I do córtex visual de duas espécies de roedores: Cavia porcellus, popularmente conhecido no Brasil como porquinho-da-índia e Rattus norvegicus, que é o rato e foi utilizada a linhagem Wistar, comumente usado nas pesquisas científicas. O porquinho-da-índia é um modelo animal muito estudado, utilizado em diversos segmentos da ciência. Apesar dessa espécie ser bem estudada, trabalhos na camada I desse animal são relativamente raros, especialmente em relação à morfologia e eletrofisiologia dos neurônios dessa região cortical. Pesquisas em ratos sobre os neurônios da camada I são mais frequentes, tanto em relação a morfologia quanto a eletrofisiologia. Para discriminar as possibilidades de diferenças na morfologia dos neurônios da camada I do córtex visual do porquinho-da-índia e do rato, este estudo classificou esses neurônios de acordo com a trajetória de seus dendritos e analisou as medidas dendríticas utilizando a técnica de injeção intracelular de biocitina. Após a classificação dos neurônios as comparações foram feitas entre os mesmos tipos celulares de cada roedor. Foram utilizados 35 porquinhos-da-índia da variedade Dunkin-Hartley de pêlo curto de ambos os sexos com idades de 4 a 5 dias de vida pós-natal. Quanto aos ratos, foram utilizados 30 ratos da variedade Wistar, de ambos os sexos com idades de 14 a 21 dias de vida pós-natal. Os animais foram anestesiados e tiveram seus encéfalos removidos, hemisférios separados e foram realizados cortes no plano coronal na região occipital onde se localiza a área visual dos roedores. As fatias foram mantidas em líquido cérebro-espinhal artificial e em seguida levadas ao microscópio para injeção de biocitina e posteriormente foram fixadas e tratadas para montagem em lâmina e contracoradas com Nissl para melhor visualização. Os neurônios encontrados foram classificados como: horizontais, ascendentes, descendentes e radias. Foram analisadas as seguintes medidas dendríticas: área do campo receptor, comprimento dendrítico total e médio, área total do corpo celular, número de dendritos, distância da pia-máter e análise da distribuição de Sholl. Dos resultados obtidos os mais notáveis foram o alcance dos ramos dendríticos e o tamanho do corpo celular dos neurônios da camada I do porquinho-da-índia quando comparados aos do rato. Isso sugere que, nessa espécie, um maior número de microcircuitos neurais podem ser estabelecidos, e por conseguinte maior taxa metabólica, justificada pelo maior tamanho do corpo celular.

The cGMP -PKG pathway is critical for inducing long-term sensitization of identified nociceptive sensory neurons

In various species, peripheral injury produces long-lasting sensitization of central and peripheral neurons representing the affected area. In Aplysia, memory-like traces (lasting days or weeks) of noxious peripheral stimulation include enhancement of central synaptic transmission and enhanced excitability of the central soma and peripheral branches of nociceptive sensory neurons. An important role for the cAMP-PKA-CREB pathway in consolidating long-term memory and inducing transcription-dependent synaptic potentiation has also been indicated by studies in rodents and Drosophila. ^ Much less attention has been paid to the cGMP-PKG pathway for transcription-dependent plasticity. Nevertheless, the cGMP-PKG pathway has been implicated in activity-dependent neural alterations lasting hours, and may trigger some forms of persistent pain. Recent evidence indicates PKG can regulate gene expression in the brain and several properties make it an attractive candidate for inducing long-term memories. ^ This dissertation reports that brief, noxious stimulation of a behaving, semi-intact preparation from mollusc, Aplysia californica, produces transcription-dependent, long-term hyperexcitability (LTH) of nociceptive sensory neurons that requires a nitric oxide (NO)-cGMP-protein kinase G (PKG) pathway and which lasts for at least 24 hours. Intracellular injection of cGMP is sufficient to induce LTH. Similarly, body wall injury induces LTH which can be blocked with specific inhibitors of the NO-cGMP-PKG pathway such as L-NMMA, ODQ, Rp-8-cGMPS, PKI-G and KT5823 by isolated perfusion of pleural ganglion sensory cells in or directly by intracellular injection. In contrast, specific inhibitors of the cAMP-PKA pathway (Rp-8-cAMPS, PKI-A and H-89) failed to block injury-induced LTH. Interestingly, co-injection of the cAMP-responsive element (CRE) blocked the induction of both cAMP and injury-induced LTH, but not cGMP-induced LTH. Furthermore, co-injection of cAMP and cGMP with the Ca2+ buffer BAPTA in reduced Ca2+ seawater blocked cAMP-, but not cGMP-induced LTH. These findings demonstrate that the NO-cGMP-PKG pathway and at least one other pathway (perhaps mediated by Ca2+), but not the cAMP-PKA pathway, are critical for inducing LTH during transient, noxious stimulation.^

Ca2+/calmodulin-binding peptides block phototransduction in Limulus ventral photoreceptors: Evidence for direct inhibition of phospholipase C

Phototransduction in Limulus photoreceptors involves a G protein-mediated activation of phospholipase C (PLC) and subsequent steps involving InsP3-mediated release of intracellular Ca2+. While exploring the role of calmodulin in this cascade, we found that intracellular injection of Ca2+/calmodulin-binding peptides (CCBPs) strongly inhibited the light response. By chemically exciting the cascade at various stages, we found the primary target of this effect was not in late stages of the cascade but rather at the level of G protein and PLC. That PLCδ1 contains a calmodulin-like structure raised the possibility that PLC might be directly affected by CCBPs. To test this possibility, in vitro experiments were conducted on purified PLC. The activity of this enzyme was strongly inhibited by CCBPs and also inhibited by calmodulin itself. Our results suggest that the calmodulin-like region of PLC has an important role in regulating this enzyme.

Brain-derived neurotrophic factor rapidly enhances synaptic transmission in hippocampal neurons via postsynaptic tyrosine kinase receptors.

Although neurotrophins are primarily associated with long-term effects on neuronal survival and differentiation, recent studies have shown that acute changes in synaptic transmission can also be produced. In the hippocampus, an area critically involved in learning and memory, we have found that brain-derived neurotrophic factor (BDNF) rapidly enhanced synaptic efficacy through a previously unreported mechanism--increased postsynaptic responsiveness via a phosphorylation-dependent pathway. Within minutes of BDNF application to cultured hippocampal neurons, spontaneous firing rate was dramatically increased, as were the frequency and amplitude of excitatory postsynaptic currents. The increased frequency of postsynaptic currents resulted from the change in presynaptic firing. However, the increased amplitude was postsynaptic in origin because it was selectively blocked by intracellular injection of the tyrosine kinase receptor (Ntrk2/TrkB) inhibitor K-252a and potentiated by injection of the phosphatase inhibitor okadaic acid. These results suggest a role for BDNF in the modulation of synaptic transmission in the hippocampus.

A study of pyramidal cell structure in the cingulate cortex of the macaque monkey with comparative notes on inferotemporal and primary visual cortex

Recent studies have revealed a marked degree of variation in the pyramidal cell phenotype in visual, somatosensory, motor and prefrontal cortical areas in the brain of different primates, which are believed to subserve specialized cortical function. In the present study we carried out comparisons of dendritic structure of layer III pyramidal cells in the anterior and posterior cingulate cortex and compared their structure with those sampled from inferotemporal cortex (IT) and the primary visual area (V1) in macaque monkeys. Cells were injected with Lucifer Yellow in flat-mounted cortical slices, and processed for a light-stable DAB reaction product. Size, branching pattern, and spine density of basal dendritic arbors was determined, and somal areas measured. We found that pyramidal cells in anterior cingulate cortex were more branched and more spinous than those in posterior cingulate cortex, and cells in both anterior and posterior cingulate were considerably larger, more branched, and more spinous than those in area V1. These data show that pyramidal cell structure differs between posterior dysgranular and anterior granular cingulate cortex, and that pyramidal neurons in cingulate cortex have different structure to those in many other cortical areas. These results provide further evidence for a parallel between structural and functional specialization in cortex.

Specialization in pyramidal cell structure in the sensory-motor cortex of the vervet monkey (Cercopethicus pygerythrus)

Recent studies have revealed systematic differences in the pyramidal cell structure between functionally related cortical areas of primates. Trends for a parallel in pyramidal cell structure and functional complexity have been reported in visual, somatosensory, motor, cingulate and prefrontal cortex in the macaque monkey cortex. These specializations in structure have been interpreted as being fundamental in determining cellular and systems function, endowing circuits in these different cortical areas with different computational power. In the present study we extend our initial finding of systematic specialization of pyramidal cell structure in sensory-motor cortex in the macaque monkey [Cereb Cortex 12 (2002) 1071] to the vervet monkey. More specifically, we investigated pyramidal cell structure in somatosensory and motor areas 1/2, 5, 7, 4 and 6. Neurones in fixed, flat-mounted, cortical slices were injected intracellularly with Lucifer Yellow and processed for a light-stable 3,3'-diaminobenzidine reaction product. The size of, number of branches in, and spine density of the basal dendritic arbors varied systematically such that there was a trend for increasing complexity in arbor structure with progression through 1/2, 5 and 7. In addition, cells in area 6 were larger, more branched, and more spinous than those in area 4. (c) 2005 IBRO. Published by Elsevier Ltd. All rights reserved.

Alterations in the phenotype of neocortical pyramidal cells in the Dyrk1A+/- mouse

The gene encoding the dual-specificity tyrosine-regulated kinase DYRK1A maps to the chromosomal segment HSA21q22.2, which lies within the Down syndrome critical region. The reduction in brain size and behavioral defects observed in mice lacking one copy of the murine homologue Dyrk1A (Dyrk1A+/-) support the idea that this kinase may be involved in monosomy 21 associated mental retardation. However, the structural basis of these behavioral defects remains unclear. In the present work, we have analyzed the microstructure of cortical circuitry in the Dyrk1A+/- mouse and control littermates by intracellular injection of Lucifer Yellow in fixed cortical tissue. We found that labeled pyramidal cells were considerably smaller, less branched and less spinous in the cortex of Dyrk1A+/- mice than in control littermates. These results suggest that Dyrk1A influences the size and complexity of pyramidal cells, and thus their capability to integrate information. (c) 2005 Elsevier Inc. All rights reserved.

Specialization in pyramidal cell structure in the sensory-motor cortex of the chacma baboon (Papio ursinus) with comparative notes on macaque and vervet monkeys

The systematic study of pyramidal cell structure has revealed new insights into specialization of the phenotype in the primate cerebral cortex. Regional specialization in the neuronal phenotype may influence patterns of connectivity and the computational abilities of the circuits they compose. The comparative study of pyramidal cells in homologous cortical areas is beginning to yield data on the evolution and development of such specialized circuitry in the primate cerebral cortex. Recently, we have focused our efforts on sensory-motor cortex. Based on our intracellular injection methodology, we have demonstrated a progressive increase in the size of, the branching structure in, and the spine density of the basal dendritic trees of pyramidal cells through somatosensory areas 3b, 1, 2, 5, and 7 in the macaque and vervet monkeys. In addition, we have shown that pyramidal cells in premotor area 6 are larger, more branched, and more spinous than those in the primary motor cortex (MI or area 4) in the macaque monkey, vervet monkey, and baboon. Here we expand the basis for comparison by studying the basal dendritic trees of layer III pyramidal cells in these same sensory-motor areas in the chacma baboon. The baboon was selected because it has a larger cerebral cortex than either the macaque or vervet monkeys; motor cortex has expanded disproportionately in these three species; and motor cortex in the baboon reportedly has differentiated to include a new cortical area not present in either the macaque or vervet monkeys. We found, as in monkeys, a progressive increase in the morphological complexity of pyramidal cells through areas 3b, 5, and 7, as well as from area 4 to area 6, suggesting that areal specialization in microcircuitry was likely to be present in a common ancestor of primates. In addition, we found subtle differences in the extent of the interareal differences in pyramidal cell structure between homologous cortical areas in the three species. (c) 2005 Wiley-Liss, Inc.