Does nerve regeneration restore normal functional patterns in mouse barrel cortex?
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Animals↗
Biomedical subjects
E Welker
Publications and source records attributed to E Welker.
Upregulation of BDNF mRNA expression in the barrel cortex of adult mice after sensory stimulation.
Upregulation of brain-derived neurotrophic factor (BDNF) mRNA expression by neuronal activity has been reported in cultured hippocampal cells and in different in vivo excitotoxic paradigms. The aim of the present study was to determine whether sensory stimulation of the whisker-to-barrel pathway alters BDNF mRNA expression in the cortex and, if so, to evaluate the specificity of this effect. To this end, a set of mystacial whiskers was unilaterally stimulated by mechanical deflection, and the expression of BDNF mRNA was analyzed in the barrel cortex by in situ hybridization (ISH) using a 35S-labeled antisense BDNF riboprobe and emulsion autoradiography. A clear-cut and specific upregulation of the BDNF mRNA expression was found at the level of the somatosensory cortex after the increased peripheral stimulation. In the barrel cortex of control mice, BDNF mRNA was present in a few cells in layers II/III and VI, whereas it was almost undetectable in layer IV. After 6 hr of whisker stimulation, increased levels of BDNF mRNA were found in layers II to VI of the contralateral barrel cortex. In layer IV, BDNF upregulation was confined to the barrels corresponding to the stimulated follicles. ISH combined with immunocytochemistry against the three calcium-binding proteins parvalbumin, calretinin, and calbindin-D28K revealed that BDNF mRNA-expressing cells do not belong to the GABAergic cell population of the barrel cortex. The present results support a role for BDNF in activity-dependent modifications of the adult cerebral cortex.
Animals↗
Membrane topology and glycosylation of the human multidrug resistance-associated protein.
The membrane topology of the human multidrug resistance-associated protein (MRP) was examined by flow cytometry phenotyping, immunoblotting, and limited proteolysis in drug-resistant human and baculovirus-infected insect cells, expressing either the glycosylated or the underglycosylated forms of this protein. Inhibition of N-linked glycosylation in human cells by tunicamycin did not inhibit the transport function or the antibody recognition of MRP, although its apparent molecular mass was reduced from 180 kDa to 150 kDa. Extracellular addition of trypsin or chymotrypsin had no effect either on the function or on the molecular mass of MRP, while in isolated membranes limited proteolysis produced three large membrane-bound fragments. These experiments and the alignment of the MRP sequence with the human cystic fibrosis transmembrane conductance regulator (CFTR) suggest that human MRP, similarly to CFTR, contains a tandem repeat of six transmembrane helices, each followed by a nucleotide binding domain, and that the C-terminal membrane-bound region is glycosylated. However, the N-terminal region of MRP contains an additional membrane-bound, glycosylated area with four or five transmembrane helices, which seems to be a characteristic feature of MRP-like ATP-binding cassette transporters.
ATP Binding Cassette Transporter, Subfamily B, Mem↗
Altered sensory processing in the somatosensory cortex of the mouse mutant barrelless.
Mice homozygous for the barrelless (brl) mutation, mapped here to chromosome 11, lack barrel-shaped arrays of cell clusters termed "barrels" in the primary somatosensory cortex. Deoxyglucose uptake demonstrated that the topology of the cortical whisker representation is nevertheless preserved. Anterograde tracers revealed a lack of spatial segregation of thalamic afferents into individual barrel territories, and single-cell recordings demonstrated a lack of temporal discrimination of center from surround information. Thus, structural segregation of thalamic inputs is not essential to generate topological order in the somatosensory cortex, but it is required for discrete spatiotemporal relay of sensory information to the cortex.
Animals↗
Altered drug-stimulated ATPase activity in mutants of the human multidrug resistance protein.
The characteristics of P-glycoprotein (MDR1), an ATP-dependent drug extrusion pump responsible for the multidrug resistance of human cancer, were investigated in an in vitro expression system. The wild-type and several mutants of the human MDR1 cDNA were engineered into recombinant baculoviruses and the mutant proteins were expressed in Sf9 insect cells. In isolated cell membrane preparations of the virus-infected cells the MDR1-dependent drug-stimulated ATPase activity, and 8-azido-ATP binding to the MDR1 protein were studied. We found that when lysines 433 and/or 1076 were replaced by methionines in the ATP-binding domains, all these mutations abolished drug-stimulated ATPase activity independent of the MgATP concentrations applied. Photoaffinity labeling with 8-azido-ATP showed that the double lysine mutant had a decreased ATP-binding affinity. In the MDR1 mutant containing a Gly185 to Val replacement we found no significant alteration in the maximum activity of the MDR1-ATPase or in its activation by verapamil and vinblastine, and this mutation did not modify the MgATP affinity or the 8-azido-ATP binding of the transporter either. However, the Gly185 to Val mutation significantly increased the stimulation of the MDR1-ATPase by colchicine and etoposide, while slightly decreasing its stimulation by vincristine. These shifts closely correspond to the effects of this mutation on the drug-resistance profile, as observed in tumor cells. These data indicate that the Sf9-baculovirus expression system for MDR1 provides an efficient tool for examining structure-function relationships and molecular characteristics of this clinically important enzyme.
ATP Binding Cassette Transporter, Subfamily B, Mem↗
Drug-stimulated ATPase activity of a deletion mutant of the human multidrug-resistance protein (MDR1).
The baculovirus-insect cell system has been used for the functional expression of the human multidrug resistance protein (MDR1) and a mutant MDR1 variant lacking a twenty amino acid segment from the first extracellular loop (delta aa78-97 MDR1). Both MDR1 proteins were found to be correctly inserted into the insect cell membrane as indicated by their interaction with MRK 16 antibody. The removal of the 78-97 segment from the first extracellular loop dramatically altered drug-stimulated ATPase activity. Rhodamine 123 or vinblastine were not able to stimulate the mutant protein and Calcein AM had also little effect. In contrast, verapamil increased the ATPase activity of the mutant almost to the same maximal level as that of the wild type. However, the verapamil concentration needed for the half maximal stimulation of the ATPase activity was found to be about hundred times higher than that for the wild type MDR1. These results indicate that a partial deletion of an extracellular loop modulates the affinity of MDR1 for its transportable substrates in a variable fashion.
ATP Binding Cassette Transporter, Subfamily B, Mem↗
Morphology and spatial distribution of corticothalamic terminals originating from the cat auditory cortex.
In this paper we studied the morphology and spatial distribution of corticothalamic axons and terminals originating from the auditory cortical fields of the cat. The anterograde tracer biocytin was injected at electrophysiologically characterized loci in the primary (AI) (N = 2), anterior (AAF) (N = 1), posterior (PAF) (N = 1) and secondary (AII) (N = 2) auditory fields. In all cases, two different types of labeled terminals were found in the auditory thalamus: small spherical endings (1-2 microns) and giant, finger-like endings (5-10 microns). After biocytin injections in AI and AAF, the majority of anterogradely labeled axons terminated in the rostral half of the pars lateralis (LV) of the ventral division of the medial geniculate body (vMGB). In LV, the corticothalamic axons ramified profusely, giving rise to dense terminal fields forming well delineated curved stripes, with small spherical endings. Additional terminal fields formed by small endings were observed in the medial division of the medial geniculate body (mMGB). Giant endings were observed in a small area in the dorsal nucleus (D) of the dorsal division of the medial geniculate body (dMGB), near its border with the vMGB. PAF projections were located in the caudal half of vMGB and in mMGB, where only small terminals were found. Giant endings were seen in the superficial part of dMGB emerging from labeled corticothalamic axons oriented in parallel to the dorsal surface of the MGB. Projections from AII gave rise to a main terminal field of small endings in D; a second terminal field consisting of giant endings intermingled with small endings was found in the deep dorsal nucleus (DD) of dMGB. We conclude that small terminals serve the feedback projection to the thalamic nucleus from which the injected cortical field receives its main input, whereas giant terminals cross the borders between the parallel ascending auditory pathways.
Acoustic Stimulation↗
Maturation of the neuronal metabolic response to vibrissa stimulation in the developing whisker-to-barrel pathway of the mouse.
We examined functional maturation in the mouse whisker-to-barrel pathway from P2 (P0 is the day of birth) to adulthood using the autoradiographic deoxyglucose (DG) method. After intraperitoneal DG injection, left whiskers C1-3 and E1 were stimulated. Sections were cut transversely through the brainstem, and coronally or tangentially through the parietal cortex. After autoradiography, the sections were stained for Nissl or for cytochrome oxidase (CO) activity. In subnuclei caudalis and interpolaris of the spinal trigeminal nucleus ipsilateral to stimulation, DG uptake evoked by the deflection of whiskers C1-3 was present at P2; in subnucleus oralis, nucleus principalis and the contralateral nucleus ventrobasalis of the thalamus, at P4; and in the contralateral barrel cortex, at P7. The first stimulus-dependent DG uptake appeared a few days after the appearance of whisker-related patterns seen in the CO- or Nissl-stained sections. In subnuclei caudalis and interpolaris, areas of stimulus-dependent DG uptake were initially larger than the CO segments representing the stimulated whiskers. Later, areas of stimulus-dependent DG uptake and CO segments matched well. DG uptake evoked by the stimulation of whisker E1 appeared 2-3 days later than that evoked by stimulation of whiskers C1-3. In nucleus principalis, one large area of stimulus-dependent DG uptake covered the representations of the caudal whiskers of all five rows--an observation made at all ages studied. In thalamus, stimulus-dependent DG uptake was found laterally in nucleus ventrobasalis. In barrel cortex, at P7, stimulus-dependent DG uptake was restricted to layers III and IV, but covered more barrels than whiskers stimulated. At P9, a second spot of high DG uptake was seen in deep layer V in register with that in layers III and IV. From P10 onwards, stimulus-dependent DG uptake stretched from layer II to layer VI, and in layer IV, in which it was highest, it was restricted to the barrels C1-3 and E1. In all stations, stimulus-dependent DG uptake decreased in magnitude after P10. While the onset of stimulus-dependent DG uptake is the result of the establishment of functional projections up to that station, the subsequent changes in size of the responding areas may well be due to the partial elimination of terminals, the maturation of local inhibitory circuits, and/or the development of cortical projections to the nuclei of termination and to the thalamic relay.
Afferent Pathways↗
The mode of activation of a barrel column: response properties of single units in the somatosensory cortex of the mouse upon whisker deflection.
Response properties of single units in the mouse barrel cortex were studied to determine the sequence in which the neurons that form a cortical column become activated by a single 'natural' stimulus. Mice (n = 11) were anaesthetized with urethane. For a total of 153 cells, grouped by cortical layer, responses to a standardized deflection of a single whisker were characterized using poststimulus time and latency histograms. Usually, for each unit, data were collected for stimulation of its principal whisker (PW; the whiskers corresponding to the barrel column in which the cell was located) and of the four whiskers surrounding the PW. In all layers, PW stimulation evoked responses at shorter latency than surround whisker stimulation. In layers II-III and IV a bimodal distribution of cells according to latency to PW stimulation was found. Statistical analysis indicated the presence of two classes of cells in each of these layers: 'fast' units (latency < 15 ms) and 'slow' units (latency > or = 15 ms). The great majority of cells in layers I, V and VI fired at latencies of > 20 ms to PW stimulation. In general, stimulation of surround whiskers evoked a smaller response than PW stimulation. The fast cells of layer IV showed the greatest response to PW stimulation (mean = 1.78 spikes/100 ms poststimulus). Their firing was maximal during the 10-20 ms poststimulus epoch, while the slow layer IV cells fired maximally during the 20-30 ms poststimulus epoch. Surround inhibition occurred in all layers within the first 10 ms after stimulus onset, during which period the fast cells are the most active ones, and are thus likely to be responsible for the surround inhibition. This notion is supported by an analysis of spike duration that showed that eight of the ten cells with a thin spike (supposed to be GABAergic; McCormick et al., J. Neurophysiol., 54, 782-806, 1985), had PW latencies of < 15 ms. We conclude that the activation of a barrel column is initially inhibitory in nature.
Action Potentials↗
The contribution of NMDA and non-NMDA receptors to fast and slow transmission of sensory information in the rat SI barrel cortex.
The main objective of this study was to establish the contribution of NMDA receptors to natural processing of somatosensory information within rat SI barrel cortex. Responses of 52 cells in layers I-IV of the rat barrel cortex were analyzed by PSTH (peristimulus histogram) analysis of evoked spikes in reply to brief deflections of the principal whisker in animals anesthetized with urethane. Short and longer peak latency responses within PSTHs were compared in the presence and absence of the specific NMDA and non-NMDA antagonists D(-)-2-amino-5-phosphonovaleric acid and 6,7-dinitroquinoxaline-2,3-dione, which were administered locally to neurons by iontophoresis and additionally tested against their putative specific agonists, NMDA and quisqualate, respectively. The results suggest the following. (1) The generation of most spikes from cells in layers I-IV is dependent upon activation of NMDA receptors. However, NMDA receptors do not contribute to responses at very short latencies commensurate with monosynaptic thalamocortical relay for layer IV cells. These appear to be entirely mediated through non-NMDA receptors. (2) In the absence of transmission through NMDA receptors, non-NMDA receptors do not generate significant spike activity in later (10-100 msec latency) discharges. (3) NMDA receptor participation in first spike generation is directly dependent upon the latency of response of the cell to principal whisker deflection. (4) Latency of response, non-NMDA receptor-mediated spike generation and laminar location were powerfully covariant. (5) In addition, it was found that cells exhibiting short-duration spikes ( < 0.7 msec; "fast-spike units") in layer IV responded powerfully at short latencies, first spikes being entirely dependent upon non-NMDA but not NMDA receptor action, later spikes (10-100 msec poststimulus) being > 80% dependent upon NMDA receptor action. It is concluded that most sensorially driven spike activity in layers I-IV is dependent upon NMDA receptor action. This appears to be enabled by contingent subthreshold depolarization largely through non-NMDA receptor action, whereas the earliest thalamocortical discharges are evoked solely through non-NMDA receptors.
2-Amino-5-phosphonovalerate↗
Barrelfield expansion after neonatal eye removal in mice.
We investigated the effect of neonatal eye removal on the tangential extent of the barrelfield in mice. Areas were measured in drawings made from tangentially cut Nisslstained sections of somatosensory cortex. We compared areas of 29 barrels, corresponding to 29 mystacial vibrissae, between adult mice enucleated at birth (n = 13) and their intact littermates (n = 13). Multivariate analysis of variance showed that the barrelfield was larger in enucleated mice. This expansion was mainly due to the increase in areal extent of the barrels corresponding to the dorsalmost row of vibrissae, and of a set of barrels corresponding to rostral vibrissae near the nose and mouth. Evidently, early enucleation has a significant cross-modal effect on the somatosensory cortex.
Aging↗
Plasticity in the barrel cortex of the adult mouse: effects of chronic stimulation upon deoxyglucose uptake in the behaving animal.
We investigated experience-dependent regulation of neuronal activity in the whisker-to-barrel pathway of the adult mouse using the autoradiographic deoxyglucose (DG) method. Animals were placed in the Lausanne whisker stimulator, and three of their whisker follicles were passively stimulated for a period of 1, 2, or 4 d. After this period, mice received a dose of DG and were placed in a cage containing a pile of wooden sticks. Mice that underwent the same procedure except the passive stimulation served as controls. Patterns of stimulus-dependent DG uptake were studied in the somatosensory cortex and in the trigeminal sensory brainstem complex. DG uptake in the barrels corresponding to the passively stimulated whiskers was lower than in controls. This decrease was present throughout the radial extent of a barrel column and was observed in all passively stimulated animals. Quantitative analysis confirmed these observations and, furthermore, showed a statistically significant decrease in DG uptake in barrels neighboring the passively stimulated ones. In half of the animals, the brainstem nuclei showed a decreased DG uptake in the representation of the passively stimulated whiskers, whereas in the other animals the pattern of DG uptake was as in controls. We propose that the signs of cortical plasticity are due to a mechanism that operates in layer IV and functions as a gate for peripheral sensory activity to enter cortical circuitry.
Animals↗
Ultrastructure of giant and small thalamic terminals of cortical origin: a study of the projections from the barrel cortex in mice using Phaseolus vulgaris leuco-agglutinin (PHA-L).
By means of tracing with the lectin Phaseolus-vulgaris leucoagglutinin (PHA-L), we examined in the thalamus of the mouse, the axon terminals of fibers originating in the barrel cortex. Vibratome sections of the brain were subjected to PHA-L immunocytochemistry and processed for light and electron microscopy. We observed small (0.5-0.8 microns in diameter) varicosities of labeled fibers in the nucleus ventrobasalis (VB) and the nucleus posterior (PO) as well as labeled giant terminals (3-5 microns in diameter) in PO. The analysis involved examination of serial sections and computer-aided reconstruction of several terminals. The small varicosities in VB appear to be small axon terminals forming distinct asymmetric synapses with small dendritic profiles. Some labeled terminals are apposed to, but not synaptically related with, the cell bodies of neurons in VB that are retrogradely labeled with PHA-L. The small varicosities seen with the light microscope in PO are terminals forming asymmetric synapses with dendritic shafts. The giant terminals in PO appear as large, vesicle-filled profiles forming part of synaptic glomeruli, i.e. complexes of one corticothalamic terminal engulfing several excrescences of a single dendrite. A giant terminal forms several asymmetric synapses (about 8) with these excrescences, as well as numerous (up to 15) puncta adhaerentia. The glomeruli are enveloped in glial lamellae, and they are often found at the bifurcations of primary dendritic segments. We suggest that the small terminals in VB are in the service of feedback signalling from the barrel cortex to its principal thalamic relay nucleus; the functional importance of this projection may reside in increased spatio-temporal discrimination. We interpret the giant terminals in PO as elements serving feed-forward processing, allowing the barrel cortex to influence, via PO, parts of the motor pathway modulating the animal's ongoing behavior.
Animals↗
Morphology of corticothalamic terminals arising from the auditory cortex of the rat: a Phaseolus vulgaris-leucoagglutinin (PHA-L) tracing study.
Phaseolus vulgaris-leucoagglutinin (PHA-L) injection in the auditory cortex of the rat labeled anterogradely corticothalamic axons whose trajectory, morphology of terminals and their distribution were analyzed in light microscopy. From the primary auditory cortex, corticofugal axons ran in a rostral direction in the white matter (external capsule), and reached the internal capsule by crossing the caudate putamen. Then, they turned caudally, crossed the reticular nucleus (RE) of the thalamus, where some of them were seen to give off collaterals, ramifying in the 'auditory sector' of RE. From RE, the parent corticofugal axons continued in a caudal and medial direction to enter in the medial geniculate body (MGB). Corticofugal axons from the auditory cortex gave rise to 2 distinct types of terminals in the thalamus. First, small boutons (about 1 micron in diameter) were observed in the ventral division of the MGB (v-MGB; the main auditory relay nucleus in the thalamus), in RE, in the lateral part of the posterior thalamic nucleus, in the dorsal division of the MGB (d-MGB), as well as occasionally in the medial division of the MGB. Giant terminals (5-10 microns in diameter) formed the second type of cortical terminals, only present in a restricted zone of the ventral portion of d-MGB. Both types of terminals were observed as boutons 'terminaux' and 'en passant'. The zone of termination in v-MGB and RE varied as a function of the site of cortical injection. The similarity in the morphology and distribution of the terminals of corticothalamic axons arising from the primary auditory cortex with those of the primary somatosensory cortex of the mouse is striking and points to the existence of a basic pattern of connectivity used in corticothalamic processing of sensory information in rodents.
Animals↗
Plasticity in the barrel cortex of the adult mouse: transient increase of GAD-immunoreactivity following sensory stimulation.
Sensory experience during perinatal life and adulthood modifies physiological and anatomical characteristics of the central nervous system. So far, this phenomenon has been studied in situations of complete or partial sensory deprivation. We here report that increased sensory stimulation, during four days, of a number of whisker follicles on the face of the adult mouse results in an increased immunoreactivity of glutamic acid decarboxylase (the biosynthetic enzyme of the inhibitory neurotransmitter GABA) in the somatosensory cortex of the adult mouse. Effects were limited to a column of tissue corresponding to the representation of the stimulated follicles and lasted two days beyond stimulation. These findings suggest that sensory stimulation transiently modifies local cortical processing.
Animals↗
Plasticity in the barrel cortex of the adult mouse: effects of peripheral deprivation on GAD-immunoreactivity.
The whisker-to-barrel pathway of the adult mouse was used in a study on the effects of peripheral sensory deprivation on GAD-immunoreactivity in the somatosensory cortex. At varying periods of time after removal of a set of vibrissal follicles, mice were processed for immunohistochemistry using an antibody against GAD. In sections tangential to the cortical surface we observed, in the barrels whose follicles were removed, decreased immunoreactivity as early as three days after surgery. The decrease was due to a lesser numerical density of stained puncta and to less intense staining of those remaining. GAD-positive somata were also less intensely stained, whereas their number did not seem to be changed. The changes, apparent at 3 days after the surgery, were restricted to the barrels corresponding to the removed follicles and were maximal at 2-4 weeks. At longer survival times (until 7 months) the immunoreactivity returned to normal, coincident with the regeneration of peripheral nerve fibres in the absence of their follicles. We conclude that GAD-immunoreactivity in the barrel cortex swiftly reacts to modifications of neuronal activity evoked in the periphery.
Afferent Pathways↗
Organization of feedback and feedforward projections of the barrel cortex: a PHA-L study in the mouse.
In order to analyze the organization of the efferent projections of single barrel columns (BC, i.e. a barrel in layer IV of parietal cortex plus the cortical tissue above and below it), we made small iontophoretic injections of the anterograde tracer Phaseolus vulgaris leucoagglutinin in the barrel cortex of 20 adult mice. On the basis of reconstructions of the sites of terminal labelling, the brain regions receiving projections from the barrel cortex could be identified and classified in five groups. Each group is characterized by the topography of the distribution of efferents arising from a single BC. The projections to the trigeminal sensory complex are point to point: i.e. one BC projects only to the site of termination of the primary sensory neurons innervating the corresponding whisker follicle. In the ventrobasal thalamic nucleus BC projections are not restricted to the corresponding barreloid; instead they contract parts of barreloids belonging to one arc. In the reticular and posterior thalamic nuclei the projections from a row of BC's converge to a collective termination site, whereas in the superior colliculus the projections from an arc of BC's converge to a common termination site. There is a complete overlap of BC projections in restricted zones within SII, motor cortex, perirhinal cortex, contralateral barrelfield, caudoputamen and pons. The organization of the efferents from the barrel cortex demonstrates a contrast between feedback and feedforward projections from this important area of neocortex.
Animals↗
Organization of the projections from barrel cortex to thalamus in mice studied with Phaseolus vulgaris-leucoagglutinin and HRP.
In order to elucidate the geometric organization of projections from the barrel cortex to the thalamus, iontophoretic injections of the anterograde tracer Phaseolus vulgaris-leucoagglutinin were made. The injections were confined to one barrel column (i.e. barrel in layer IV + cortical tissue above and below it). Axonal terminations could be demonstrated in three thalamic nuclei: reticularis (RT), ventrobasalis (VB) and posterior (PO). Anterograde terminal labelling was obtained in RT + VB; in PO only; or in RT + VB + PO. The terminals labelled in PO were much larger than those in RT and VB. The termination areas in RT, VB and PO were shaped like rods which have a rostro-caudal orientation. These cortico-thalamic projections are discretely and topographically organized. The clearest such arrangement was found in VB. Here, projections from the A row of barrels in BF terminate dorsally, whereas those from the C row end ventrally. Barrel A1 projects to the lateral part of VB, whereas A4, to more medial parts; other rows are arranged similarly. These results were compared with the distribution of thalamo-cortical projection neurons that were labelled after iontophoretic HRP injections in individual barrels. We concluded that the corticothalamic projections originating from one barrel column contact an are of barreloids in VB.
Animals↗