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J DeFelipe

Publications and source records attributed to J DeFelipe.

At least 37 records · Page 2Linked to original sources
PubMed2000

Colocalization of glutamate ionotropic receptor subunits in the human temporal neocortex.

alpha-Amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA), kainate and N-methyl-D-aspartate (NMDA) receptors represent major classes of glutamate receptors (GluR) which play fundamental roles in normal excitatory synaptic activity and, probably, in the etiology of several brain diseases. These receptors are composed of multiple receptor subunit proteins, and the differential expression of these subunits in cortical neurons is considered to be one of the substrates for the functional diversity of cortical excitatory circuitry. In the monkey neocortex, different subpopulations of neurons have been identified on the basis of immunocytochemical colocalization studies using subunit-specific antibodies, but no comparable investigations have been made in the human neocortex. The aim of the present study was to determine quantitatively GluR subunit combinations in the human temporal neocortex by double-labeling immunocyto- chemical experiments. We quantified the neuronal populations expressing different receptor subtypes with fluorescent tags visualizing them with confocal laser microscopy. We studied AMPA, kainate- and NMDA-receptor subunits, using antibodies against GluR1, GluR2, GluR2/3, GluR2/4, GluR5/6/7 and NMDAR1 subunits. A high degree of colocalization (93-100%) using combinations of antibodies against GluR2 with GluR2/3, GluR2/3 with GluR2/4, and GluR2 or GluR2/4 with NMDAR1 was found, whereas for other combinations the degree of colocalization varied between 38% and 88%. Some of the percentages reported here are similar to those found in the monkey cortex, whereas others differ considerably. These results emphasize the diversity of excitatory circuits in the human neocortex, and suggest species differences with regard to some of these GluR-mediated circuits.

Animals↗
PubMed1999

Variation in the spatial relationship between parvalbumin immunoreactive interneurones and pyramidal neurones in rat somatosensory cortex.

The morphology of pyramidal neurones was revealed by intracellular injection of Lucifer Yellow (LY) in fixed tangential cortical slices taken from rat primary somatosensory cortex. Slices were processed with a combination of antibodies to allow visualization of both the LY-injected neurones and parvalbumin immunoreactive (PV-ir) cell bodies, by confocal microscopy. Basal dendritic fields of pyramidal neurones in layer V were larger and more complex than those of layer III. Furthermore, the number of PV-ir cell bodies contained within the basal dendritic territories of pyramidal neurones in layer V was significantly greater than in layer III (mean +/- s.e.m., 36.3 +/- 3.0 and 20.9 +/- 1.6, respectively). These findings have functional implications both in terms of physiological characteristics, and inhibitory modulation of receptive field properties, of cortical neurones.

Animals↗
PubMed1999

Transport of CSF antibodies to Galpha subunits across neural membranes requires binding to the target protein and protein kinase C activity.

In the light of functional studies, it has been suggested that antibodies directed to alpha subunits of G-proteins delivered into cerebrospinal fluid (CSF) reached and blocked the function of neural transducer proteins. Current understanding indicates that IgGs do not move freely across plasma membranes. Therefore, to characterize the uptake of these antibodies by neural cells, anti-Gi2alpha IgGs were labeled with 125I, fluorescein or with gold particles. The expression of Galpha subunits was also reduced by blocking their mRNA with antisense oligodeoxynucleotides (ODN). Following intracerebroventricular (icv) injection of gold-conjugated anti-Gi2alpha IgGs, electrondense particles entered and became distributed in the cytoplasm and plasma membranes of neural cells. Scattered particles were also found in dendrites and nuclei. Unlabeled IgGs diminished cerebral signals of fluorescein-labeled anti-Galpha IgGs, indicating that this uptake can be saturated. Cerebral radiostaining promoted by in vivo anti-Gi2alpha 125I-IgGs was almost absent in Gi2alpha knocked-down mice, but not after decreasing the quantity of Gzalpha subunits. The immunosignals of CSF anti-Galpha 125I-IgGs, as well as the impairment of opioid-evoked antinociception, were increased by agonist-induced activation of G protein-coupled receptors. The impairing effect of the antibodies on opioid-evoked antinociception was prevented by agents blocking the cellular uptake of proteins, i.e., cytochalasin B, BSA, DMSO, H7, and by down regulation of protein kinase Cbeta1 (PKCbeta1). In mice treated with an ODN to PKCbeta1 mRNA, 125I-IgGs to Gi2alpha subunits remained bound to periventricular structures and did not spread to deeper areas of the CNS. These results indicate that IgGs delivered into the CSF show a saturable binding to Galpha subunits that translocate to the external side of the neural membrane before being internalized by a PKCbeta1-dependent mechanism.

Analgesics↗
PubMed1999

Myr+-Gi2 alpha and Go alpha subunits restore the efficacy of opioids, clonidine and neurotensin giving rise to antinociception in G-protein knock-down mice.

In mice whose Gi/o-protein function had been impaired by antisense 'knock-down' or pertussis toxin treatment, i.c.v. injection of myr+-Gi/o alpha subunits restored the effectiveness of beta-endorphin, morphine, DPDPE, clonidine and neurotensin to produce antinociception. Myr+-G alpha subunits of the class of G-proteins actually impaired were more effective than unlike but related myr+-G alpha subunits. Selectivity was noted in that only exogenous myr+-G alpha subunits affected (enhanced) the activity of agonists in G alpha-deficient signalling systems. This treatment had little effect on agonist potency when the impairment resided at the receptor level. The potential of the opioids, clonidine and R-PIA to increase G alpha-related in vitro hydrolysis of GTP was also re-established after injecting myr+-Gi2 alpha subunits into Gi2-knocked-down mice. Myr+-Gi2 alpha subunits pre-incubated with GTPgammaS or GDPbetaS before i.c.v. injection did not improve the activity of agonists in vivo (antinociception) or in vitro (regulation of low Km GTPase). After impairing the function of PKCbeta1 by antisense treatment or with the inhibitor H7, the effect of myr+-G alpha subunits on agonist potency was prevented. Electron microscope analysis showed the entry of gold-conjugated myr+-G alpha subunits into neural cells. These particles were found in the cytoplasm, associated with the plasma membranes of different neuronal processes and also in synaptic junctions. In cultured neurons and astrocytes myr+-Gi2 alpha-associated fluorescence was internalised in a dose-dependent manner and distributed in the plasma membrane and cytosol, as well as in nuclei of dividing astrocytes. Thus, G alpha subunits in CSF enter into neurons and functionally couple to the receptor-triggered signalling cascade. As G-proteins have been implicated in the pathophysiology of several neural disorders, this finding may be valuable in the therapy of such dysfunctions.

Analgesics↗
PubMed1999

Chandelier cells and epilepsy.

The main goal of this article is to review certain aspects of the circuitry of the human cerebral cortex that may be particularly relevant for the development, maintenance or spread of seizures. There are a number of different structural abnormalities that are commonly found in the cortex of epileptic patients, but these abnormalities do not appear to be intrinsically epileptogenic, since some patients displaying them are epileptic (after variable delays) whereas others are not. Therefore, cortical circuits in an affected brain may undergo a series of changes that finally cause epilepsy. In this article, it is proposed that the chandelier cell, which is considered to be the most powerful cortical GABAergic inhibitory interneuron, is probably a key component of cortical circuits in the establishment of human intractable temporal lobe epilepsy. These cells (among other types) have been found to be lost or reduced at epileptic foci in both experimental animals and epileptic patients. A hypothesis is presented by which the normal variability in the number of interneurons might explain the predisposition of some individuals to develop epilepsy more than others as a result of a lesion or other precipitating factors that lead to loss of neurons. The sources of GABAergic input on dendrites and somata of cortical pyramidal cells originate from many and diverse types of interneurons but, at the level of the axon initial segment of these cells, all synapses come from a few chandelier cells (five or less). Loss of one class of interneurons ending on soma and dendrites might have relatively little impact on the inhibitory control of the pyramidal cell. However, if chandelier cells were affected, it would have serious consequences for the inhibitory control of the pyramidal cells. Evidence suggests that the loss of chandelier cells may be non-specific and that when this occurs epilepsy may develop. Therefore, these cells might represent a key component in the aetiology of human temporal lobe epilepsy.

Animals↗
PubMed1998

Chandelier cell axons are immunoreactive for GAT-1 in the human neocortex.

We have examined the pattern of immunostaining for the high-affinity GABA transporter GAT-1 in the human temporal neocortex. Immunocytochemistry for GAT-1 labels terminal-like puncta in the neuropil and around unstained cell bodies. The characteristic terminal portions of chandelier cell axons (Ch-terminals, which form multiple inhibitory GABAergic synaptic contacts with the axon initial segments of pyramidal cells) were among the strongest immunocytochemically stained elements for GAT-1. Since Ch-terminals are immunoreactive for the calcium-binding protein parvalbumin, experiments were carried out to study the co-localization of GAT-1 and parvalbumin in Ch-terminals. These experiments showed that the vast majority of Ch-terminals immunoreactive for GAT-1 were also immunoreactive for PV. We concluded that GAT-1 transporter may have an important functional role in controlling pyramidal cell activity.

Adult↗
PubMed1998

Local changes in GTP-binding protein immunoreactivities in human epileptogenic neocortex.

The relative levels of guanine nucleotide-binding protein alpha-subunits Gi1alpha, Gi2alpha, Gi3alpha, Go(alpha), Gs(alpha), and Gx/z(alpha) were measured in neocortex removed at surgery from patients with intractable temporal lobe epilepsy. Immunoreactivity was quantified using specific polyclonal antisera against the Galpha-subunits according to the Laurell "rocket" immunoelectrophoresis technique. We compared the G protein contents of spiking (active) and nonspiking (nonactive) cortical regions, based on intraoperative electrocorticography, within the same and different patients. There were no clear trends for lower or higher levels of G-protein subtypes to be found in the samples of protein extracts from nonspiking regions as compared to spiking regions. However, comparison of paired samples of spiking and nonspiking cortex within the same patient demonstrated that levels of certain G-protein subtypes were either increased or decreased in all patients. This indicates that cortical regions with enhanced neuronal activity may produce microzonal alterations in the levels of G proteins. Moreover, our results suggest that high levels of Gi1alpha and low levels of the other G-protein subtypes appear to be associated with a greater susceptibility to maintaining spiking activity.

Adult↗
PubMed1997

Synaptic connections of calretinin-immunoreactive neurons in the human neocortex.

Previous immunocytochemical studies in the cerebral cortex of various species have shown that the calcium-binding protein calretinin (CR) labels specific subpopulations of nonspiny nonpyramidal cells (interneurons). The present study attempts to characterize morphologically and chemically the microcircuitry of CR-immunoreactive (CR-ir) neurons in the human temporal neocortex. Postembedding immunocytochemistry for CR and GABA and combination immunocytochemistry for CR and nonphosphorylated neurofilament protein (NPNFP) or for CR and the calcium-binding proteins parvalbumin (PV) and calbindin (CB) showed CR multiterminal endings frequently innervating the distal apical dendrite or the cell body and proximal dendrites of NPNFP-ir or CB-ir pyramidal cells, respectively. Cell bodies of interneurons immunoreactive for CB or PV were innervated only occasionally by CR multiterminal endings, whereas certain GABA neurons were surrounded by them. Furthermore, CR-ir axon terminals formed either symmetrical (the majority) or asymmetrical synapses with a variety of postsynaptic elements. These results indicate that different subpopulations of CR interneurons exist that are specialized for selective innervation of somatic or dendritic regions of certain pyramidal and nonpyramidal neurons.

Adult↗
PubMed1997

Altered synaptic circuitry in the human temporal neocortex removed from epileptic patients.

Quantitative electron microscopic methods were used to study possible alterations in presumptive excitatory and inhibitory synaptic circuits in human neocortex removed from patients with intractable temporal lobe epilepsy. Synaptic density was compared between normal and abnormal regions as identified by Nissl staining and immunocytochemistry for the Ca2+-binding protein parvalbumin (PV). The normal regions showed a normal cytoarchitecture and normal pattern of staining for PV, whereas the abnormal regions displayed focal neuronal cell loss and a decrease in immunostaining for PV. In the abnormal regions the overall synaptic density (per 100 microm2 and per mm3) was approximately 30% higher than in normal regions, which corresponded to an increase of approximately 300 million synapses per mm3. The number of excitatory and inhibitory synapses was significantly higher and lower, respectively, than in normal regions. We suggest that these changes are a result of a focal sprouting of excitatory axon terminals and loss of inhibitory terminals which leads to hyperexcitatory synaptic circuits. These circuits may represent a neural substrate for the initiation or propagation of seizure activity in human epileptogenic neocortex.

Adolescent↗
PubMed1997

Loss of inhibitory synapses on the soma and axon initial segment of pyramidal cells in human epileptic peritumoural neocortex: implications for epilepsy.

The peritumoural neocortex removed from epileptic patients represents an important region for research because of its possible relationship to the generation, maintenance, and propagation of seizures. The peritumoural neocortex removed from an epileptic patient showing a regrowth of an anaplastic astrocytoma was examined in detail using immunocytochemistry for gamma-aminobutyric acid, glutamic acid decarboxylase, parvalbumin, nonphosphorylated neurofilament protein, glial fibrillary acidic protein, and histocompatibility antigen HLA-DR. The patterns of immunostaining were compared with the cytoarchitecture and myeloarchitecture in adjacent sections, and with the patterns of immunostaining observed in normal control neocortex. Furthermore, quantitative electron microscopy was used to compare the synaptic densities of presumptive excitatory and inhibitory synapses between regions showing different grades of cytoarchitectural and neurochemical alterations in the peritumoural neocortex, and to compare these regions with normal neocortex. A variety of changes in synaptic circuits in the peritumoural neocortex was found, but it appears that neurons within the less abnormal-looking regions were involved in altered synaptic circuits that might contribute to epileptic activity. In these regions, the most prominent change was the loss of inhibitory synapses on the soma and axon initial segment of pyramidal cells, but numerous excitatory synapses were present on their dendrites that would make these neurons hyperexcitable. However, the most abnormal regions histologically were likely a primary zone for progression of the tumour, with many surviving neurones, but which received and formed very few synapses; thus, they were probably unrelated to the initiation, maintenance, or propagation of seizures.

Adult↗
PubMed1997

Colocalization of parvalbumin and calbindin D-28k in neurons including chandelier cells of the human temporal neocortex.

Chandelier cells are cortical GABAergic interneurons with a unique synaptic specificity enabling them to exert a strong inhibitory influence on pyramidal cells. By using immunocytochemistry for the calcium-binding protein calbindin D-28k in the human temporal neocortex, we have found numerous immunoreactive processes that were identified as chandelier cell axon terminals. This was a striking find since in previous immunocytochemical studies of the primate neocortex, chandelier cell axon terminals had been shown to be immunoreactive for another calcium-binding protein, parvalbumin, and colocalization studies indicate that parvalbumin and calbindin are present in almost completely separate neuronal populations. Here, we present double-label immunofluorescence experiments showing that parvalbumin and calbindin immunoreactivities are colocalized in certain neurons that include a subpopulation of chandelier cells whose cell bodies are located mainly in layers V and VI of the human temporal neocortex. The results suggest a selective laminar distribution of neurochemical subtypes of chandelier cells which is a peculiar feature of the organization of the human neocortex.

Adolescent↗
PubMed1997

Double bouquet cell axons in the human temporal neocortex: relationship to bundles of myelinated axons and colocalization of calretinin and calbindin D-28k immunoreactivities.

We have examined the distribution of double bouquet cell axons, immunocytochemically stained for the calcium-binding proteins calretinin and calbindin D-28k in the human temporal neocortex, in relation to bundles of myelinated axons (originating from pyramidal cells) and the colocalization of these calcium-binding proteins. The large number and regularity of distribution of double bouquet cell axons was clearly visualized in tangential sections from cortical layers III--V. In these sections, we estimated that the mean number +/- standard deviation of double bouquet cell axons per 10,000 microns2 was 11.65 +/- 0.44 with a mean diameter of 12.10 +/- 0.63 microns and a mean center-to-center spacing of 29.8 +/- 0.91 microns. These values are very similar to those previously reported in the monkey neocortex. The distribution of double bouquet cell axons was closely related to bundles of myelinated axons; there was overlapping with basically a one-to-one correspondence. Finally, double-label immunofluorescence experiments revealed that the vast majority of double bouquet cell axons immunoreactive for calbindin were also stained for calretinin. Since relatively few cell somata were double-labeled in the human temporal cortex, we concluded that double bouquet cells may represent a significant subpopulation of neurons that colocalize these calcium-binding proteins.

Adult↗
PubMed1997

Types of neurons, synaptic connections and chemical characteristics of cells immunoreactive for calbindin-D28K, parvalbumin and calretinin in the neocortex.

This article provides a general account of types of neurons, synaptic connections and chemical characteristics (colocalization studies) of cells immunoreactive for the three main calcium-binding proteins found in the neocortex, namely, calbindin-D28K, parvalbumin and calretinin. The main conclusion is two-fold. First, all, or the majority, of calbindin-, parvalbumin- and calretinin-immunoreactive cells are smooth nonpyramidal neurons (interneurons) which participate in a variety of complex cortical circuits that may differ depending on the species, cortical area or layer where they are located. Second, in general, different types of nonpyramidal neurons are stained for each of these calcium-binding proteins and display different chemical characteristics regarding a variety of neurotransmitters (or related compounds), cell surface markers and receptors. However, a certain overlap exits, which also shows regional and species differences.

Adult↗
PubMed1996

Colocalization of calbindin D-28k, calretinin, and GABA immunoreactivities in neurons of the human temporal cortex.

The calcium-binding proteins calbindin D-28k (CalB) and calretinin (CalR) have been shown to be useful markers of neuronal subpopulations located mainly in layers II-III of the neocortex of a variety of species, including human. Double labeling immunocytochemical studies of CalB, CalR, and GABA in experimental animals have shown that CalB and CalR are present in separate subpopulations of neurons. However, there are no studies of colocalization of these calcium-binding proteins and GABA in the human neocortex. The principal goal of the present work was to investigate the degree of colocalization of these substances in layers II-III of the human temporal neocortex, using a postembedding immunocytochemical method. The patterns of staining for CalB, CalR, and GABA in the human cortex were similar to those found in monkey neocortex. However, the degree of colocalization for certain combinations was different from that reported in the monkey and other experimental animals. A relatively large proportion of CalB- and CalR-immunoreactive cells (approximately 71% and 74%, respectively) were found to be immunoreactive for GABA. However, the degree of colocalization of CalB with CalR was low (between 4% and 6%). Thus, our quantitative and qualitative data suggest that these calcium-binding proteins are present in similar cortical circuits in all primates, but that in the human neocortex, there might be additional GABAergic and perhaps also non-GABAergic interneurons with unique chemical characteristics.

Adolescent↗
PubMed1996

Distribution of parvalbumin immunoreactivity in the neocortex of hypothyroid adult rats.

Early hypothyroidism produces a generalized damage in the brain and in particular, changes in the connectivity of neocortical sensory areas. In this paper, the potential alterations in local neocortical circuits have been explored using immunocytochemistry for parvalbumin (PV) in normal and hypothyroid adult rats. The number and radial distribution of PV-positive cells were similar in both groups, but in hypothyroid rats, the density of PV-positive terminal-like puncta and processes was dramatically reduced, especially in layers II-III and the lower part of layers IV-V and VI. These results suggest that thyroid hormones are necessary for normal development of cortical circuits in which PV-positive cells are involved.

Animals↗
PubMed1996

Inhibitory neurons in the human epileptogenic temporal neocortex. An immunocytochemical study.

Immunocytochemical methods were used to study alterations in inhibitory neuronal circuits in human neocortex resected during surgical treatment of intractable temporal epilepsy associated or not with brain tumours. The epileptogenic cortex was characterized and divided into spiking or non-spiking zones by intraoperative electrocorticography (ECOG). The resected cortex was cut into blocks, sectioned and stained immunocytochemically for visualization of glutamic acid decarboxylase (GAD), the calcium-binding protein, parvalbumin (PV) and glial fibrillary acidic protein (GFAP). A variety of alterations in cortical neuronal circuits as revealed by immunocytochemical and histological methods were found. Similar alterations in inhibitory neuronal circuits appear to occur independently of the primary epileptogenic site and pathology associated with epilepsy, which suggests that there is possibly a common basic underlying mechanism that leads to seizure activity. These changes were apparently unrelated to ECOG findings at surgery, which bring into question the value of the use of interictal epileptic discharges recorded by ECOG to guide cortical resections. The most conspicuous and common change was the loss of chandelier cells. The finding that these cells are among the most vulnerable types of GABAergic interneurons in the epileptogenic temporal cortex indicates that they might be of great functional importance, since the axon terminals of chandelier cells are likely to exert powerful regulation of impulse generation in cortical pyramidal cells. Therefore, these cells might represent a key component in the aetiology of human epilepsy.

Adolescent↗
PubMed1995

A light and electron microscopic study of calbindin D-28k immunoreactive double bouquet cells in the human temporal cortex.

Correlative light and electron microscopic methods were used to examine the morphology, distribution and synaptic connections of double bouquet cells immunoreactive for the calcium-binding protein calbindin D-28k in the human temporal neocortex. Double bouquet cells form symmetric synapses with small dendritic shafts and dendritic spines. The distribution and proportion of synapses found in the present work are very similar to those found in previous studies on the synaptic connectivity of double bouquet cells in the monkey cerebral cortex. Thus, double bouquet cells are probably involved in similar synaptic circuits in monkeys and humans.

Adult↗