Sodium channels in dendrites of rat cortical pyramidal neurons.

The voltage-dependent properties that have been directly demonstrated in Purkinje cell and hippocampal pyramidal cell dendrites play an important role in the integrative capacities of these neurons. By contrast, the properties of neocortical pyramidal cell dendritic membranes have been more difficult to assess. Active dendritic conductances near sites of synaptic input would have an important effect on the input-output characteristics of these neurons. In the experiments reported here, we obtained direct evidence for the existence of voltage-dependent Na+ channels on the dendrites of neocortical neurons by using cell-attached patch and whole cell recordings from acutely isolated rat neocortical pyramidal cells. The qualitative and quantitative properties of dendritic and somatic currents were indistinguishable. Insofar as Na+ currents are concerned, the soma and primary apical dendrite can be considered as one relatively uniform compartment. Similar dendritic Na+ currents on dendrites in mature neurons would play an important role in determining the integrative properties of these cortical units.     

Modeling back propagating action potential in weakly excitable dendrites of neocortical pyramidal cells.

Simultaneous recordings from the soma and apical dendrite of layer V neocortical pyramidal cells of young rats show that, for any location of current input, an evoked action potential (AP) always starts at the axon and then propagates actively, but decrementally, backward into the dendrites. This back-propagating AP is supported by a low density (-gNa = approximately 4 mS/cm2) of rapidly inactivating voltage-dependent Na+ channels in the soma and the apical dendrite. Investigation of detailed, biophysically constrained, models of reconstructed pyramidal cells shows the following. (i) The initiation of the AP first in the axon cannot be explained solely by morphological considerations; the axon must be more excitable than the soma and dendrites. (ii) The minimal Na+ channel density in the axon that fully accounts for the experimental results is about 20-times that of the soma. If -gNa in the axon hillock and initial segment is the same as in the soma [as recently suggested by Colbert and Johnston [Colbert, C. M. & Johnston, D. (1995) Soc. Neurosci. Abstr. 21, 684.2]], then -gNa in the more distal axonal regions is required to be about 40-times that of the soma. (iii) A backward propagating AP in weakly excitable dendrites can be modulated in a graded manner by background synaptic activity. The functional role of weakly excitable dendrites and a more excitable axon for forward synaptic integration and for backward, global, communication between the axon and the dendrites is discussed.     

Ectopic dendrites occur only on cortical pyramidal cells containing elevated GM2 ganglioside in alpha-mannosidosis.

In a variety of neuronal storage diseases, cortical pyramidal cells elaborate ectopic dendrites at the axon hillock. A feature common to all the diseases characterized by ectopic dendrites is an elevated level of GM2 ganglioside in cerebral cortex. In cats with one such disease, alpha-mannosidosis, the number of pyramidal cells bearing ectopic dendrites is small; the present study shows that GM2 ganglioside is stored only in those pyramidal neurons exhibiting ectopic dendrites. Using a Golgi-electron microscopy method with periodic acid-Schiff (PAS) staining, we first established that pyramidal cells bearing ectopic dendrites contained PAS+ membranous inclusions, consistent with storage of glycolipids. In contrast, those with smooth axon hillocks accumulated PAS- floccular inclusions, consistent with storage of oligosaccharides. Next, application of a monoclonal antibody against GM2 ganglioside revealed that subsets of both pyramidal and intrinsic neurons contained GM2-like immunoreactivity. Every GM2+ cell contained PAS+ membranous inclusions, indicating that pyramidal cells bearing ectopic dendrites stored GM2 ganglioside. In cats with alpha-mannosidosis induced by swainsonine, some pyramidal neurons showed GM2-like immunoreactivity after 4 weeks of treatment, whereas ectopic dendrites only became evident after 7 weeks of treatment. Thus, GM2 ganglioside accumulated in pyramidal neurons before ectopic dendrites emerged from the axon hillock. We propose that the reinitiation of dendrite growth on mature pyramidal cells is brought about by accumulated GM2 ganglioside.     

Host cell invasion by the opportunistic pathogen Toxoplasma gondii.

Toxoplasma gondii is an obligate intracellular protozoan that infects an astonishing variety of vertebrate hosts including humans. Classified in the phylum Apicomplexa, T. gondii causes an opportunistic disease, toxoplasmosis, in individuals with immune dysfunction and congenital disease in infected infants. Re-emergence of toxoplasmosis as a life-threatening disease in patients with AIDS is anticipated in the wake of emerging multi-drug resistant strains of HIV. In immunodeficient patients, the available evidence suggests that tissue pathology associated with T. gondii infection is due to parasite-directed lytic destruction of individual host cells. The Toxoplasma lytic cycle begins when the parasite actively invades a target cell. In association with invasion, T. gondii sequentially discharges three sets of secretory organelles beginning with the micronemes, which contain adhesive proteins involved in parasite attachment to a host cell. Deployed as protein complexes, several micronemal proteins possess vertebrate-derived adhesive sequences that function in binding receptors on the surface of a target cell. Each protein in these adhesive complexes fulfills a specific role in movement through the secretory pathway, targeting to the micronemes, or adhesion. It is anticipated that these adhesive complexes recognize a variety of host receptors, including some that are expressed on multiple cell types, and that this diversity in host cell receptors contributes to the remarkably broad tissue- and host-range of T. gondii.     

Quantification of in vivo tumor invasion and vascularization by computerized image analysis

The matrix-inserted surface transplantation model is an in vivo assay used to analyse the kinetics of tumor-vessel interactions during different stages of skin carcinoma progression. This system allows the study of host-tumor interface, i.e. penetration of tumor cells into normal host tissue as well as infiltration of normal host cells into the tumor. In the present study, image analysis algorithms for processing and quantifying the extent of such migratory and tissue remodeling events are presented. The proposed method is non-parametric and its originality lies in its particularity to take into account the specific geometry of tumor-host interface. This methodology is validated by evaluating the contribution of matrix metalloproteases (MMPs) in skin carcinoma invasion and vascularization through pharmacological and genetic approaches.     

Emergence of functional subnetworks in layer 2/3 cortex induced by sequential spikes in vivo

During cortical circuit development in the mammalian brain, groups of excitatory neurons that receive similar sensory information form microcircuits. However, cellular mechanisms underlying cortical microcircuit development remain poorly understood. Here we implemented combined two-photon imaging and photolysis in vivo to monitor and manipulate neuronal activities to study the processes underlying activity-dependent circuit changes. We found that repeated triggering of spike trains in a randomly chosen group of layer 2/3 pyramidal neurons in the somatosensory cortex triggered long-term plasticity of circuits (LTPc), resulting in the increased probability that the selected neurons would fire when action potentials of individual neurons in the group were evoked. Significant firing pattern changes were observed more frequently in the selected group of neurons than in neighboring control neurons, and the induction was dependent on the time interval between spikes, N-methyl-D-aspartate (NMDA) receptor activation, and Calcium/calmodulin-dependent protein kinase II (CaMKII) activation. In addition, LTPc was associated with an increase of activity from a portion of neighboring neurons with different probabilities. Thus, our results demonstrate that the formation of functional microcircuits requires broad network changes and that its directionality is nonrandom, which may be a general feature of cortical circuit assembly in the mammalian cortex.Layer 2/3 neurons in the barrel cortex play a central role in integrative cortical processing (1–4). Neurons in layer 2/3 are interconnected with each other, and their axons and dendrites traverse adjacent barrel areas (5, 6). Recent calcium (Ca²⁺) imaging studies in awake animals showed that two very closely localized layer 2/3 pyramidal neurons are independently activated by different whiskers (7). In addition, adjacent layer 2/3 neurons have different receptive field properties; signals from different whiskers may emerge on different spines in the same neurons (8, 9). These findings suggest that the organization of functional subnetworks in somatosensory layer 2/3 is heterogeneous at the single-cell level and that microcircuits are assembled at a very fine scale (10). In vivo whole-cell recording experiments have also shown that most, but not all, layer 2/3 pyramidal neurons receive subthreshold depolarization by single-whisker stimulation with much broader receptive fields than neurons in layer 4 (11, 12). These anatomical and functional data suggest that electric signals relayed to the cortex by whisker activation are greatly intermingled within layer 2/3 neurons, and that studying the mechanisms by which these layer 2/3 neurons make connections may be critical for understanding the cortical network organizing principles underlying somatosensation.A previous modeling study suggested that spike timing-dependent plasticity (STDP) can lead to the formation of functional cortical columns and activity-dependent reorganization of neural circuits (13–16). However, how spikes arising in multiple neurons in vivo influence their connectivity is poorly understood. In this study using two-photon glutamate photolysis, which allowed us to control neuronal activity in a spatially and temporally precise manner, we examined activity-dependent cellular mechanisms during network rearrangement generated by repetitive spike trains in a group of neurons. We found that repetitive spikes on a group of neurons induced the probability of the neurons firing together. This circuit plasticity required spiking at short intervals among neurons and is expressed by N-methyl-D-aspartate (NMDA) receptor- and Calcium/calmodulin-dependent protein kinase II (CaMKII)-dependent long-lasting connectivity changes. The probability of firing was differentially affected by the order of the spike sequence but was not dependent on the physical distance between neurons. Thus, our data show that neuronal connectivity within a functional subnetwork is established in not only a preferred but also a directional manner.     

Aniracetam reduces glutamate receptor desensitization and slows the decay of fast excitatory synaptic currents in the hippocampus.

Aniracetam is a nootropic drug that has been shown to selectively enhance quisqualate receptor-mediated responses in Xenopus oocytes injected with brain mRNA and in hippocampal pyramidal cells [Ito, I., Tanabe, S., Kohda, A. & Sugiyama, H. (1990) J. Physiol. (London) 424, 533-544]. We have used patch clamp recording techniques in hippocampal slices to elucidate the mechanism for this selective action. We find that aniracetam enhances glutamate-evoked currents in whole-cell recordings and, in outside-out patches, strongly reduces glutamate receptor desensitization. In addition, aniracetam selectively prolongs the time course and increases the peak amplitude of fast synaptic currents. These findings indicate that aniracetam slows the kinetics of fast synaptic transmission and are consistent with the proposal [Trussell, L. O. & Fischbach, G. D. (1989) Neuron 3, 209-218; Tang, C.-M., Dichter, M. & Morad, M. (1989) Science 243, 1474-1477] that receptor desensitization governs the strength of fast excitatory synaptic transmission in the brain.     

Impulse flow dependency of galanin release in vivo in the rat ventral hippocampus.

Using microdialysis and a sensitive RIA, we have studied the in vivo release of the neuropeptide galanin (GAL) from the ventral hippocampus of freely moving rats. The spontaneous outflow of GAL-like immunoreactivity (GAL-LI) (1.8 +/- 0.3 fmol per ml per 20 min) was dependent on the presence of extracellular Ca2+ and was inhibited by tetrodotoxin. Evoked release induced by infusion of KCl (60 mM) or veratridine (148 microM) was also Ca(2+)-dependent and sensitive to tetrodotoxin. Electrical stimulation of the ventral limb of the diagonal band nuclei induced a frequency-dependent (50-200 Hz) and tetrodotoxin-sensitive overflow of GAL-LI in the hippocampus. In vitro GAL-LI release (1.0 +/- 0.02 fmol per ml per 5 min), studied in slices of rat ventral hippocampus, was also Ca(2+)-dependent and was increased in a concentration-dependent manner by KCl depolarization. This study demonstrates the release of the neuropeptide GAL in the rat central nervous system. The in vivo release is related to the activity of the cholinergic GAL-LI-containing cells in the septal diagonal band nuclei. The results are discussed in relation to the coexistence of GAL and acetylcholine within the septal/diagonal band complex.     

Immunoreactivity of calcium binding protein secretagogin in the human hippocampus is restricted to pyramidal neurons

Disturbed calcium homeostasis plays a crucial role in the aetiology of Alzheimer's disease (AD) and the aging process. We evaluated immunoreactivity of secretagogin, a recently cloned calcium binding protein, in hippocampus and adjacent entorhinal cortex of 30 neuropathologically examined post mortem brains (m:f=12:18; mean age, 79.8+/-15.1 years). The study group consisted of 15 cases fulfilling the criteria for high probability of AD according to the NIA-Reagan Institute Criteria and 15 cases with no to medium probability. Sections were incubated with secretagogin-specific antibodies and the number of immunoreactive neurons as well as staining intensities in both neurons and neuropil were assessed. Both cellular and neuropil immunoreactivity were restricted to subiculum and Ammons horn. Cellular immunoreactivity was further restricted to pyramidal neurons and showed a hierarchical distribution: the mean percentage of immunoreactive neurons was highest in sector CA3 (64.41%), followed by CA2 (44.09%), CA4 (34.38%), CA1 (10.9%), and the subiculum (2.92%; P<0.001, except CA2-CA4, P>0.05), while it did not differ significantly between groups with different degrees of AD pathology. The pattern of secretagogin immunoreactivity resembles that of calcium sensor proteins as it is restricted to a subset of neurons and therefore secretagogin could serve highly specialized tasks in neuronal calcium signalling.     

2型糖尿病小鼠海马锥体细胞及毛细血管电镜观察

目的 研究2型糖尿病模型db/db小鼠海马锥体细胞、毛细血管超微结构变化。方法 糖尿病组：6周龄C57BL／KsJ(db db )小鼠5只。对照组：非糖尿病小鼠C57BL／KsJ(?/ )5只。于30周龄时,灌注固定取脑、透射电镜下观察海马CA1区。结果 电镜下可见糖尿病小鼠海马锥体细胞凋亡、毛细血管基底膜增厚。结论 如上改变可能是糖尿病认知功能 减退的病理基础。     

Antidepressants increase glucocorticoid and mineralocorticoid receptor mRNA expression in rat hippocampus in vivo.

Adrenal corticosteroids bind to hippocampal glucocorticoid (GR) and mineralocorticoid receptors (MR), thereby affecting neurochemical transmission leading to altered mood, behaviour and neuroendocrine control. Serotoninergic (5-HT) and noradrenergic projections innervate the hippocampus, interacting with corticosteroid-sensitive cells. We have previously demonstrated that lesions of 5-HT neurons reduce hippocampal GR and MR mRNA levels and now examine whether acute or chronic treatment with antidepressant drugs, which potentiate endogenous monoamines by inhibiting their reuptake, affect hippocampal GR and MR mRNA expression in vivo. Rats were treated with amitriptyline (20 mg/kg.day-1), desipramine (10 mg/kg.day-1) or citalopram (20 mg/kg.day-1). After 2 or 14 days animals were killed, RNA extracted and GR and MR mRNA expression quantified by slot blot hybridization. Amitriptyline for 2 days led to a significant increase in MR (by 23 +/- 6%, compared with saline-treated controls), but not GR, mRNA expression. After 14 days amitriptyline, expression of both MR (87 +/- 27% rise) and GR mRNA (56 +/- 18% rise) had increased significantly in hippocampus, but not in parietal cortex. Desipramine for 14 days also increased MR (60 +/- 9%) and GR (42 +/- 9%) mRNA expression, though 14 days of citalopram altered only MR mRNA expression (17 +/- 5%). Thus, antidepressant drug administration elevates MR and GR mRNA expression in hippocampus, but not parietal cortex, in a time-dependent manner.     

Mammalian cell invasion by closely related Trypanosoma species T. dionisii and T. cruzi

Protozoan parasites of the genus Trypanosoma can infect virtually all mammalian species. Within this genus, Trypanosoma dionisii from bats and Trypanosoma cruzi that causes Chagas' disease, belonging to the subgenus Schizotrypanum, can invade mammalian cells. The mechanisms of cell invasion by T. dionisii are poorly understood. To address that question, metacyclic trypomastigotes (MT) and human epithelial HeLa cells were used. Similarly to genetically divergent T. cruzi strains G (TcI) and CL (TcVI), associated, respectively with marsupial and human infections, T. dionisii infectivity increased under nutritional stress, a condition that induces host cell lysosome exocytosis required for parasite internalization. For efficient internalization, T. dionisii depended on MT protein tyrosine kinase (PTK) and Ca(2+) mobilization from acidocalcisomes, whereas T. cruzi strains also relied on phosphatidylinositol 3-kinase (PI3K), protein kinase C (PKC) and Ca(2+) released from thapsigargin-sensitive compartments. T. dionisii-induced signaling in host cells implicated PKC and Ca(2+) mobilized from thapsigargin-sensitive stores, like T. cruzi, but without PI3K involvement. Unlike T. cruzi, T. dionisii metacyclic forms did not use l-proline as source of energy required for internalization. Molecules related to T. cruzi surface glycoproteins involved in MT-host cell interaction were undetectable in T. dionisii. The difference in the surface profile of the two species was also inferred from the susceptibility of T. dionisii metacyclic forms to complement-mediated lysis, as opposed to complete resistance of T. cruzi. In summary, the two Trypanosoma species display distinct surface profiles but invade host cells through a common mechanism involving lysosome mobilization to the site of parasite entry.     

Glial cell loss in the hippocampus of alcoholics

Alcohol abuse is often accompanied by cognitive impairment. Memory and concentration problems, especially, are described in neuropsychologically tested abstinent alcoholics. This impairment seems to be reversible, because a neuropsychological study has reported that after >5 years of abstinence, former alcoholics performed normally compared with a control group. The neuropathological correlate to the cognitive impairment associated with alcoholism is still under debate. Hippocampus is an area of the brain primarily involved in learning and memory, and the hippocampus is affected in several demential diseases. Over recent years, it has become evident that the glial cells in the brain, especially the astrocytes, play a central part in many activities that are critical to the normal functions of the brain. The total number of neuron and glial cells in the hippocampus was estimated in five severely affected alcoholics and five controls with an unbiased stereological technique: the optical fractionator. A statistically significant loss of 37% of the glial cells was found globally in the hippocampus of the alcoholics compared with controls. A reduction of astrocytes and oligodendrocytes and, to a lesser degree, microglial cells accounted for this loss. No loss of neurons was found in the hippocampus from alcoholics. The loss of glial cells and the potential for regeneration of these cells after cessation of alcohol abuse is discussed in the light of the results of clinical studies of former alcoholics.     

Calcium electrogenesis in distal apical dendrites of layer 5 pyramidal cells at a critical frequency of back-propagating action potentials

Action potentials in juvenile and adult rat layer-5 neocortical pyramidal neurons can be initiated at both axonal and distal sites of the apical dendrite. However, little is known about the interaction between these two initiation sites. Here, we report that layer 5 pyramidal neurons are very sensitive to a critical frequency of back-propagating action potentials varying between 60 and 200 Hz in different neurons. Bursts of four to five back-propagating action potentials above the critical frequency elicited large regenerative potentials in the distal dendritic initiation zone. The critical frequency had a very narrow range (10-20 Hz), and the dendritic regenerative activity led to further depolarization at the soma. The dendritic frequency sensitivity was suppressed by blockers of voltage-gated calcium channels, and also by synaptically mediated inhibition. Calcium-fluorescence imaging revealed that the site of largest transient increase in intracellular calcium above the critical frequency was located 400-700 micrometer from the soma at the site for initiation of calcium action potentials. Thus, the distal dendritic initiation zone can interact with the axonal initiation zone, even when inputs to the neuron are restricted to regions close to the soma, if the output of the neuron exceeds a critical frequency.     

On the formation of gamma-coherent cell assemblies by oriens lacunosum-moleculare interneurons in the hippocampus

Gamma frequency (30-80 Hz) network oscillations have been observed in the hippocampus during several behavioral paradigms in which they are often modulated by a theta frequency (4-12 Hz) oscillation. Interneurons of the hippocampus have been shown to be crucially involved in rhythms generation, and several subtypes with distinct anatomy and physiology have been described. In particular, the oriens lacunosum-moleculare (O-LM) interneurons were shown to synapse on distal apical dendrites of pyramidal cells and to spike preferentially at theta frequency, even in the presence of gamma-field oscillations. O-LM cells have also recently been shown to present higher axonal ramification in the longitudinal axis of the hippocampus. By using a hippocampal network model composed of pyramidal cells and two types of interneurons (O-LM and basket cells), we show here that the O-LM interneurons lead to gamma coherence between anatomically distinct cell modules. We thus propose that this could be a mechanism for coupling longitudinally distant cells excited by entorhinal cortex inputs into gamma-coherent assemblies.     

T cell immunosenescence in vitro and in vivo.

The term "immunosenescence" refers to an age-associated dysregulation of immune function which contributes to the increased susceptibility of the elderly to infectious disease. Although there are age-associated changes measurable in the innate immune system (Pawelec et al., 1998c), it is the adaptive, particularly T cell, system which is most susceptible to the deleterious effects of aging. In this minireview, characteristics of aging in long-term human T cell cultures will be summarized, and the parallels between the in vitro model and in vivo immunosenescence will be documented. The use of culture models to screen for ways of manipulating immunosenescence in vitro may provide a basis for intervention to ameliorate immunosenescence in vivo.     

NogoA restricts synaptic plasticity in the adult hippocampus on a fast time scale

Whereas the role of NogoA in limiting axonal fiber growth and regeneration following an injury of the mammalian central nervous system (CNS) is well known, its physiological functions in the mature uninjured CNS are less well characterized. NogoA is mainly expressed by oligodendrocytes, but also by subpopulations of neurons, in particular in plastic regions of the CNS, e.g., in the hippocampus where it is found at synaptic sites. We analyzed synaptic transmission as well as long-term synaptic plasticity (long-term potentiation, LTP) in the presence of function blocking anti-NogoA or anti-Nogo receptor (NgR) antibodies and in NogoA KO mice. Whereas baseline synaptic transmission, short-term plasticity and long-term depression were not affected by either approach, long-term potentiation was significantly increased following NogoA or NgR1 neutralization. Synaptic potentiation thus seems to be restricted by NogoA. Surprisingly, synaptic weakening was not affected by interfering with NogoA signaling. Mechanistically of interest is the observation that by blockade of the GABA(A) receptors normal synaptic strengthening reoccurred in the absence of NogoA signaling. The present results show a unique role of NogoA expressed in the adult hippocampus in restricting physiological synaptic plasticity on a very fast time scale. NogoA could thus serve as an important negative regulator of functional and structural plasticity in mature neuronal networks.     

Invasion of human follicular thyroid carcinoma cells in an in vivo invasion model.

Models that demonstrate histological invasion of extracellular matrix barriers by tumor cell lines are useful for assessing new methods to treat or prevent tumor metastasis. An in vivo invasion model using acellular human dermal matrix has been described in a murine squamous cell carcinoma line. The present study examined the application of this tumor invasion model to another epithelial cell line derived from a different species. A human follicular thyroid carcinoma cell line, known to be invasive by other assays, was grown on the dermal-epidermal basement membrane surface of human acellular dermal matrix in culture and then grafted in athymic mice. Immunohistochemical staining of type IV collagen was used to identify the basement membrane and invasion was determined as penetration of the basement membrane by tumor cells. Identification of the human tumor cells in the in vivo grafts was done by in situ hybridization with species specific probes. FTC-133 tumor cells did not invade the matrix after 4 weeks of growth in in vitro culture, but there was extensive loss of the basement membrane and infiltration of the tumor cells into the dermis after 2 weeks growth in vivo. This study suggests that the in vivo dermal matrix model of invasion is applicable to a broad range of epithelial carcinoma cell lines to study their capability to penetrate basement membrane. A model such as this may be useful for studying the local effects of genetic manipulations of implanted tumor cell populations, leading to the development of therapeutic agents that block invasion.     

Multibranch activity in basal and tuft dendrites during firing of layer 5 cortical neurons in vivo

Layer 5 pyramidal neurons process information from multiple cortical layers to provide a major output of cortex. Because of technical limitations it has remained unclear how these cells integrate widespread synaptic inputs located in distantly separated basal and tuft dendrites. Here, we obtained in vivo two-photon calcium imaging recordings from the entire dendritic field of layer 5 motor cortex neurons. We demonstrate that during subthreshold activity, basal and tuft dendrites exhibit spatially localized, small-amplitude calcium transients reflecting afferent synaptic inputs. During action potential firing, calcium signals in basal dendrites are linearly related to spike activity, whereas calcium signals in the tuft occur unreliably. However, in both dendritic compartments, spike-associated calcium signals were uniformly distributed throughout all branches. Thus, our data support a model of widespread, multibranch integration with a direct impact by basal dendrites and only a partial contribution on output signaling by the tuft.