Bacterial colonization of microbial biofilms in oral squamous cell carcinoma.

OBJECTIVES: The purpose of this prospective clinical study was to identify the bacterial spectra on the surface of oral squamous cell carcinomas (OSCC) in comparison to oral mucosa of patients with a higher risk to emerge an OSCC and a control group to determine their susceptibility to various common antibiotics. MATERIAL AND METHODS: Swabs from 90 patients, 30 patients of each group, were cultured on media for aerobes and anaerobes and tested with agar diffusion and Etest. RESULTS: The predominant pathogens of the normal healthy oral mucosa were aerobes. The ratio between aerobes and anaerobes was 2:1, balanced in risk patients and inverted in the OSCC group. Altogether, 1,006 isolates were cultured. The most frequent strains were 47 viridans streptococci, 30 Staphylococcus species, 14 Enterococcus faecalis, 36 Neisseria species, 14 Escherichia coli, and 23 other aerobes, 66 Peptostreptococcus species, 39 Fusobacterium species, and 34 Prevotella species. The resistance rates in the OSCC group were penicillin 40%, ampicillin 57%, doxycycline 23%, clindamycin 47%, and amoxicillin/clavulanic acid 20%, but up to 100% of pathogens were susceptible to azithromycin, telithromycin, levofloxacin, and moxifloxacin. CONCLUSION: Gram-negative anaerobes play a decisive role in the development of postoperative infections in patients with OSCC. This tumor special type of colonization does not agree with the normal flora of the oral cavity. CLINICAL RELEVANCE: Biofilms on OSCC surfaces provide an important reservoir for anaerobic bacteria. As a consequence, a proposal for an antibiotic prophylactic regime should be given.

Kainic acid induces sprouting of retinal neurons.

The neurotoxin kainic acid caused dose-dependent morphological changes in horizontal cells of the retinas of adult cats and rabbits. High concentrations of kainic acid killed the cells, but when exposed to sublethal doses they contracted their dendritic fields and sent sprouting processes into the inner retina. It appears that kainic acid can induce neuronal growth as well as degeneration and that the potential for morphological plasticity is still present in neurons of the adult mammalian retina.

Pharmacological analysis of cortical circuitry.

The cortical circuitry of the visual cortex has been worked out in great detail. Anatomical investigations reveal stereotyped connections within cortical columns and specific long-range connections between distant columns. Pharmacological techniques for blocking the activity in individual cortical layers or columns allow the microdissection of the cortical circuit. These studies could relate specific functional roles to particular cortical connections.

Reconstructing cortical connections in a dish.

During development of the cortex, efferent projection neurons located in distinct cortical layers send their axons to different targets, and afferent fibers establish connections with cortical target cells of a particular layer. Recent studies have shown that layer- and cell-specific afferent and efferent cortical connections established in culture are similar to those observed in vivo. The results of these experiments provide evidence for the existence of diffusible and membrane-bound guidance factors for specific sets of axons. Furthermore, they suggest the use of different molecules to navigate axons towards their target, regulate target innervation and mediate target cell recognition.

Cortical circuitry in a dish.

In the mammalian neocortex, local connections as well as projections to and from the cortex are organized in cortical layers. Recent studies have demonstrated that the formation of the patterns of afferent and efferent cortical connections in organotypic co-cultures are similar to those observed in vivo. These findings provide some insights into the cellular strategies that operate during the development of layer-specific cortical connections.

Spatial distributions of guidance molecules regulate chemorepulsion and chemoattraction of growth cones.

It is generally assumed that gradients of chemotropic molecules are instrumental to the wiring of the nervous system. Recently, two members of the secreted class III semaphorin protein family have been implicated as repulsive (Sema3A) and attractive (Sema3C) guidance molecules for cortical axons (). Here, we show that stabilized gradients of increasing semaphorin concentrations elicit stereotyped responses from cortical growth cones, independent of the absolute concentration and the slope of these gradients. In contrast, neither repulsive effects of Sema3A nor attractive effects of Sema3C were observed when axons were growing toward decreasing semaphorin concentrations. Thus, growth cone guidance by gradients of chemotropic molecules is robust and reproducible, because it is primarily independent of the exact dimensions of the gradients.

Semaphorin 3A-vascular endothelial growth factor-165 balance mediates migration and apoptosis of neural progenitor cells by the recruitment of shared receptor.

The dynamic and coordinated interaction between cells and their microenvironment controls cell migration, proliferation, and apoptosis, mediated by different cell surface molecules. We have studied the response of a neuroectodermal progenitor cell line, Dev, to a guidance molecule, semaphorin 3A (Sema3A), described previously as a repellent-collapsing signal for axons, and we have shown that Sema3A acts as a repellent guidance cue for migrating progenitor cells and, on prolonged application, induces apoptosis. Both repulsion and induction of cell death are mediated by neuropilin-1, the ligand-binding component of the Sema3A receptor. The vascular endothelial growth factor, VEGF165, antagonizes Sema3A-induced apoptosis and promotes cell survival, migration, and proliferation. Surprisingly, repulsion by Sema3A also depends on expression of VEGFR1, a VEGF165 receptor, expressed in Dev cells. Moreover, we found that these repulsive effects of Sema3A require tyrosine kinase activity, which can be attributed to VEGFR1. These results indicate that the balance between guidance molecules and angiogenic factors can modulate the migration, apoptosis (or survival), and proliferation of neural progenitor cells through shared receptors.

Neurogenesis in the adult dentate gyrus after cortical infarcts: effects of infarct location, N-methyl-D-aspartate receptor blockade and anti-inflammatory treatment.

Stimulation of cell proliferation and neurogenesis in the adult dentate gyrus has been observed after focal and global brain ischemia but only little is known about the underlying mechanisms. We here analyzed neurogenesis in the dentate gyrus after small cortical infarcts leaving the hippocampal formation and subcortical regions intact. Using the photothrombosis model in adult rats, focal ischemic infarcts were induced in different cortical areas (sensorimotor forelimb and hindlimb cortex) and proliferating cells were labeled at days 3-14 after infarct induction with bromodeoxyuridine. At 2, 4, and 10 weeks after ischemia, immunocytochemistry was performed with immature neuronal (doublecortin), mature neuronal (neuronal nuclei antigen) and glial (calcium-binding protein beta S100beta) markers. When compared with sham-operated controls, animals with infarcts in the forelimb as well as hindlimb cortex revealed an increase in survival of newborn progenitor cells at four and 10 weeks after the insult with predominance at the ipsilateral side. Triple immunofluorescence and confocal laser scanning microscopy revealed an increase in neurogenesis in all groups that was more pronounced 10 weeks after the infarct. Application of the N-methyl-D-aspartate (NMDA)-receptor antagonist MK-801 during lesion induction significantly enhanced neurogenesis in the dentate gyrus. An even stronger increase in newborn neurons was observed after anti-inflammatory treatment with indomethacine during the first 16 days of the experiment. The present study demonstrates that small cortical infarcts leaving subcortical structures intact increase neurogenesis in the dentate gyrus and that these processes can be stimulated by N-methyl-D-aspartate receptor blockade and anti-inflammatory treatment.

Controllability of an aversive stimulus in depressed patients and health controls: a study using slow brain potentials.

Slow brain potentials (CNV and PINV) were recorded from 18 patients with primary depression and 18 health controls under three experimental conditions. A short tone (S1) indicated the occurrence of an aversive tone stimulus (S2) which could not be terminated by a motor reaction in Condition 1 (C1) but was terminated in C2. In C3 the reaction time (RT) had to be faster than the shortest mean RT from C1 and C2 in order to stop the aversive stimulus. For both groups the RT decreased from C1 to C2 to C3 and the CNV increased from C2 to C3. CNV and RT did not differ between the groups in either condition. In C1 and C3 (uncontrollability and restricted control over the aversive stimulus) patients developed a marked PINV which was not observed in the control group. In view of other investigations which found a PINV in normal subjects in situations of sudden, unexpected uncontrollability, the present results demonstrate that depressed patients are more sensitive to levels and variation of uncontrollability than healthy subjects. A possible relationship to Seligman's concept of helplessness is discussed.

Relationships between dendritic morphology and cytochrome oxidase compartments in monkey striate cortex.

In primate striate cortex, staining for the mitochondrial enzyme cytochrome oxidase reveals a regular pattern of intense staining, the blobs, which are surrounded by the lighter stained interblob regions. Neurons in both compartments exhibit profound functional differences: blob cells have color selective, unoriented receptive fields, whereas interblob cells are usually not color selective and have oriented receptive fields. Neuroanatomical tracing studies have shown that blob and interblob cells receive different inputs and participate in different projections. It is not known, however, whether this compartmental organization is also reflected in the dendritic morphology of individual cells. We therefore combined intracellular staining with cytochrome oxidase histochemistry to study the relationship between cell morphology and blob pattern in layers 2 and 3 of macaque striate cortex. Single cells were injected with the fluorescent dye lucifer yellow in lightly fixed tangential sections. Adjacent sections were reacted for cytochrome oxidase to reveal the blobs. The spatial relationship between stained cells and the pattern of the blobs were subsequently determined by aligning the sections by using radially running blood vessels as landmarks. Our results show that pyramidal cells located in blob and interblob regions do not differ in their soma size, spine density, and basal dendritic field structure. This indicates that the characteristic functional properties of the neurons in both compartments do not depend on the morphology of their dendritic trees. Since the elongation of the dendritic fields of blob and interblob cells was also found to be similar, we conclude that cortical orientation selectivity is not generated through elongated dendritic fields. We found several cells with dendrites freely crossing the borders between blob and interblob regions. These cells might correspond to cells with "mixed" receptive field properties, e.g., color selective oriented cells, which in physiological studies were found at the transition from blob to interblob regions. However, there were also a number of cells that respected the borders. A quantitative analysis of the dendritic fields revealed that 67% of the cells located close to the borders have a tendency to confine their dendrites to only one compartment. Thus the pattern of basal dendrites of these cells might be shaped by the parcellation of the striate cortex in blob and interblob regions. These dendritic field asymmetries may help to maintain the segregation at the single cell level into different processing channels in monkey striate cortex.

Morphological types of projection neurons in layer 5 of cat visual cortex.

Pyramidal cells in layer 5 of the visual cortex have multiple cortical and subcortical projection sites. Previous studies found that many cells possess bifurcating axons and innervate more than one cortical or subcortical target, but cells projecting to both cortical and subcortical targets were not observed. The present study examines the morphology of cells in cat visual cortex projecting to the superior colliculus, the main subcortical target of layer 5, and cells in layer 5 projecting to cortical areas 18 and 19. The neurons that give rise to these different projections were retrogradely labelled and intracellularly stained in living brain slices. Our results show that cells within each projection group have several morphological features in common. All corticotectal cells have a long apical dendrite forming a large terminal tuft in layer 1. Their cell bodies are medium sized to large, and their basal dendrites form a dense and symmetrical dendritic field. Corticocortical cells in layer 5 have a very different morphology: their apical dendrites are short and they never reach higher than layers 2/3. Their cells bodies are small to medium sized and they have fewer basal dendrites than corticotectal cells. Thus there are two morphologically distinct projection systems in layer 5, one projecting to cortical and the other one to subcortical targets, suggesting that these two systems transmit different information from the visual cortex. Among the corticotectal cells with the largest cell bodies we found some cells whose basal and apical dendrites were almost devoid of spines. Spiny and spinefree corticotectal cells also have different intrinsic axon collaterals and therefore play different roles in the cortical circuitry. While many spiny corticotectal cells have axon collaterals that project to layer 6, spinefree corticotectal cells have fewer axon collaterals and these do not arborize in layer 6. We suggest that the two morphological types of corticotectal cells might be related to functional differences known to exist among these cells. We discuss how the presence or absence of spines affects the integration of the synaptic input and how this might be related to the cells' functional properties.

GABAA receptor alpha 1 subunit, an early marker for area specification in developing rat cerebral cortex.

Changes in the expression of neurotransmitter receptors in developing cerebral cortex may be related to the functional maturation of distinct areas. In the present study, we have tested whether GABAA receptor expression in neonatal rats reflects the differentiation of cortical areas. Specifically, the alpha 1 subunit, one of the most prevalent GABAA receptor subunits in adult cerebral cortex, is up-regulated postnatally, suggesting a link with the establishment of inhibitory circuits. Using immunohistochemistry with a subunit-specific antiserum, we observed a striking area- and lamina-specific increase in staining for GABAA receptors containing the alpha 1 subunit (alpha 1-GABAA receptors), from low levels in neonates to an intense and uniform staining in adults. Already at birth, the alpha 1-subunit immunoreactivity selectively demarcated the boundaries of certain cortical areas. In particular, the primary somatosensory (S1) and visual (V1) areas were distinctly delineated with a band of alpha 1-subunit immunoreactivity located in the developing layers III and IV. The staining ended abruptly at the presumptive boundaries of S1 and V1, adjacent areas being unstained at this age. Around postnatal day 3, clusters of alpha 1-subunit positive cells were seen in layers III-IV of S1 and V1 extending their dendrites up to layer I, where they arborized profusely. In addition, the distribution of alpha 1-GABAA receptors in S1 revealed in detail the differentiation of the barrel field during early postnatal development. Although staining was observed in all areas by postnatal day 6, differences in the laminar distribution of alpha 1-GABAA receptors persisted for at least 1 more week. Our results provide evidence for the existence of area-specific boundaries in neocortex of newborn rats before layers III-IV are fully differentiated and innervated by cortical afferents. Furthermore, the area- and lamina-specific maturation of alpha 1-GABAA receptor staining demonstrates the value of this marker for investigating the cytoarchitectonic differentiation of cortical areas during development.

Pharmacological modulation of on and off ganglion cells in the cat retina.

We studied the effects of 2-amino-4-phosphonobutyric acid and 2-amino-4-phosphonovaleric acid, which are antagonists to excitatory amino acids, on brisk-sustained(X) and brisk-transient(Y) ganglion cells in the cat retina. The ganglion cells were recorded extracellularly with a multibarreled electrode in the intact eye in vivo and drugs were applied iontophoretically. We found contrasting effects of 2-amino-4-phosphonobutyric acid and 2-amino-4-phosphonovaleric acid on ON- and OFF-centre cells irrespective of the brisk-sustained(X)/brisk-transient(Y) dichotomy. The maintained discharge and the light response of ON-centre cells decreased during 2-amino-4-phosphonobutyric acid and 2-amino-4-phosphonovaleric acid application, whereas the maintained discharge of OFF-centre cells increased. The light response of OFF-centre cells was not significantly affected. 2-Amino-4-phosphonovaleric acid was generally less potent than 2-amino-4-phosphonobutyric acid. It was shown in the mudpuppy retina [Slaughter and Miller (1981) Science, N.Y. 211, 182-185; Slaughter and Miller (1981) Invest. ophthal. Vis. Sci. 20, 44] that 2-amino-4-phosphonobutyric and 2-amino-4-phosphonovaleric acid selectively block the response of ON-bipolar cells. Assuming that these drugs act on the same synaptic sites in the cat retina, one expects a block of ON-centre ganglion cells. Moreover, the drug response of OFF-centre ganglion cells is then consistent with Sterling's hypothesis [Sterling (1983) A. Rev. Neurosci. 6, 149-185] that OFF-ganglion cells receive a direct inhibitory input from ON-bipolars. For some period 19 out of 108 cells responded to 2-amino-4-phosphonobutyric acid and 2-amino-4-phosphonovaleric acid in an opposite way to that described above: ON-centre cells increased and OFF-centre cells decreased their maintained discharge. It is proposed that this "reverse response" may be mediated by autoreceptors to excitatory amino acids or due to different receptor sites triggering antagonistic response mechanisms.

Cholecystokinin in the cat retina. Action of exogenous CCK8 and localization of cholecystokinin-like immunoreactivity.

The effects of applying cholecystokinin (CCK8) iontophoretically onto cat retinal ganglion cells were studied in the optically intact eye of the cat. CCK8 suppressed both the maintained and the light evoked discharge of brisk ganglion cells, irrespective of their being on- or off-center, brisk-transient, or brisk-sustained and independent of the state of light adaptation. The inhibitory action of CCK8 in the cat retina is opposite from its excitatory action in other parts of the brain. Using an antiserum to cholecystokinin, immunoreactivity was localized in horizontal cells and amacrine cells of the cat retina. Inconsistently immunoreactivity also was found in ganglion cells and fibers.

Specification of layer-specific connections in the developing cortex.

One of the basic tasks of neurobiology is to understand how the precision and specificity of neuronal connections is achieved during development. In this paper we reviewed some recent in vitro studies on the developing mammalian cerebral cortex that have been made towards this end. The results of these experiments provided evidence that membrane-associated molecules are instrumental for the formation of specific afferent and efferent cortical projections. Substrate-bound molecules guide growing axons towards their target, regulate the timing of thalamocortical innervation and mediate target cell recognition. Moreover, a newly described glycoprotein, defined by a monoclonal antibody, revealed a molecular heterogeneity in the developing white matter. Since this molecule has opposite effects on thalamic and cortical axons, it might play a role in the segregation of axons running to and from the cortex. Substrate-bound cues are important during the formation of local cortical circuits. In vitro assays demonstrated that molecular components confined to individual cortical layers control the laminar specificity of cortical axon branching. This suggests that similar developmental strategies contribute to the laminar specification of extrinsic and intrinsic cortical circuits. Thus substrate-bound molecules might provide the framework for subsequent activity-dependent mechanisms that control the elaboration of precise connections between the cortical columns. A major challenge ahead is to identify the factors that mediate these processes and to determine their mode of action. Recently, two families of proteins, the netrins and the semaphorins/collapsins, have been identified as growth cone signals in the developing spinal cord (reviewed in Goodman, 1994; Colamarino and Tessier-Lavigne, 1995a; Dodd and Schuchardt, 1995; Kennedy and Tessier-Lavigne, 1995). Semaphorins/collapsins appear to regulate axonal guidance by repelling growth cones and by inhibiting axonal branching and synapse formation. Originally, netrins have been purified as diffusible chemoattractants for commissural axons of the dorsal spinal cord, but it is now well established that they can also function as chemorepellent factors for other classes of neurons. Since netrins are related to extracellular matrix components and since they can bind to the cell surface, they might also act as local guidance cues. A possible role of netrins and semaphorins/collapsins in the development of cortical connections is likely to be resolved in the near future. The identification of the factors that regulate specific branching patterns of cortical neurons might provide a better understanding of cortical development, but it might also be relevant to some aspects of plasticity and repair in the adult cortex.

Non-Hebbian synapses in rat visual cortex.

In the mammalian CNS, long term potentiation can be induced by repeatedly pairing presynaptic stimulation with postsynaptic depolarization of a single cell, similar to a model proposed by Hebb, that synaptic strengthening occurs as a result of correlated pre- and postsynaptic activity. However, our experiments indicate that the Hebbian rule is not strictly valid in the cortex. Double intracellular recordings showed that synaptic reinforcement is not confined to the depolarized postsynaptic neuron, but is also observed in adjacent but not coactivated neurons. The enhancement and its spread is stimulus-specific, it occurs only for fibres stimulated during the pairing procedure. During development, this spread might lead to a characteristic organizing principle of the cortex, the clustering of cells with similar functional properties.

Morphology of identified projection neurons in layer 5 of rat visual cortex.

We studied the morphology of neurons in layer 5 of rat visual cortex (area 17) projecting to the contralateral hemisphere and the superior colliculus. Double labelling with fluorescent tracers indicated that these projections arise from different populations of cells. To reveal the morphology of the cells we stained retrogradely labelled neurons intracellularly in living brain slices. Callosal projecting pyramidal cells have 3-6 basal dendrites and an apical dendrite which never reaches higher than layer 3. Corticotectal cells have 6-8 basal dendrites and a prominent apical dendrite which always forms a large tuft in layer 1. Thus, neurons in the same cortical layer that give rise to different projections also differ in their morphology. However, each population of neurons has a rather stereotyped dendritic branching pattern, despite a large variation in soma size.

Development of vasoactive intestinal polypeptide (VIP)-containing neurons in organotypic slice cultures from rat visual cortex.

Using immunohistochemistry we have been studying the postnatal maturation of vasoactive intestinal polypeptide (VIP)-positive neurons in organotypic slice cultures from rat visual cortex. The development in vitro is compared with the occurrence of VIP-containing cells in vivo, where they are first observed around postnatal day 5. A further increase in number and morphological maturation occurs within the following 3 weeks. In cultures prepared from 1- or 2-day-old rats, i.e. before VIP is expressed in vivo, VIP-containing neurons appear after about 5 days and gradually increase in number over the next 2 weeks. Thus the time course of postnatal expression of VIP in vitro and the morphology of VIP-immunoreactive neurons in culture closely matches the situation in vivo. These observations suggest that the maturation of VIP-containing neurons occurs independently of cortical afferents and that the intrinsic connectivity and activity is sufficient for their postnatal maturation. Therefore organotypic slice cultures should be a suitable system to study mechanisms of neurochemical maturation in the cortex.

Cortical barrelfields in organotypic slice cultures from rat somatosensory cortex.

We are attempting to determine the factors that influence both the formation and maintenance of barrels in the rodent somatosensory cortex. Cytochrome oxidase histochemistry and Nissl staining were used to identify the presence of barrels in slice cultures of rat cortex. Barrels were observed in sagittally cut slices, in cortical layer IV, from animals aged postnatal day 5-10 that were cultured for at least 10 days. No differences were observed in the barrel pattern from animals at different postnatal ages, or from cortex that was cultured for different lengths of time. In contrast, cortical barrels in tangentially cut sections from similarly aged animals were clearly visible after one day in culture, but they disappeared after two days in culture. These results suggest that intact connections within cortical columns are sufficient to sustain the functional architecture of the somatosensory cortex in vitro.

Localization of aspartate aminotransferase and cytochrome oxidase in the cat retina.

Using immunohistochemical techniques, we demonstrate aspartate aminotransferase (AAT)-like immunoreactivity in cone pedicles and ganglion cells of the cat retina. An identical pattern was seen when we stained for cytochrome oxidase activity, a marker for neurons which have a high metabolic activity. Tetrodotoxin selectively blocked the cytochrome oxidase labeling of ganglion cells. AAT is a key enzyme in the metabolism of aspartate and glutamate and has been proposed as a marker for neurons which use aspartate/glutamate as a neurotransmitter. Due to the close correlation between AAT-like immunoreactivity and cytochrome oxidase activity, we suggest that, at least in the retina, AAT-like immunoreactivity in fact labels cells which have a high metabolic activity.