Clermont Beaulieu

Quantitative aspects of synaptogenesis in the rat barrel field cortex with special reference to GABA circuitry

The postnatal establishment of cortical connectivity was studied by estimating the number (numerical density, synapse‐to‐neuron ratio, and total number) of the overall synaptic population and its distribution into gamma‐aminobutyric acid (GABA)‐immunopositive and GABA‐immunonegative synaptic contacts in the developing rat somatosensory cortex. These numerical data were obtained using the unbiased disector method in combination with GABA postembedding immunocytochemistry. The numerical density of both synaptic populations was low in the early postnatal period (postnatal days 5 and 10, P5, P10) after which it abruptly increased between P10 and P15 to approach adult values. However, since cortical volume continues to increase after this age, the number of synapses per neuron and the total number of synapses reached adult values only by P30. There was no evidence of overproduction of either GABA or non‐GABA synapses. Direct comparison between the two synaptic populations revealed a similar developmental pattern with the exception of the period around P20 when the production of GABA synapses slowed down. Thus, while the formation of non‐GABA synapses proceeded in a continuous manner throughout the first month of life, GABA synapse production was accomplished in two consecutive waves. We suggest that the second delayed wave of GABA synapse formation is related to the great developmental plasticity of the cortical inhibitory system.

Axonal sprouting of CA1 pyramidal cells in hyperexcitable hippocampal slices of kainate-treated rats

CA1 pyramidal cells become hyperexcitable following hippocampal kainate lesions. To examine if axonal sprouting contributes to this epileptiform activity, the local axonal arborization of CA1 pyramidal cells was examined after intracellular labelling with biocytin in hippocampal slices from control rats and in hyperexcitable slices obtained from rats treated with kainate (bilateral intracerebroventricular injections) 2‐4 weeks previously. Biocytin‐labelled cells with an axon that could be followed from the soma to the alveus were drawn and reconstructed with a camera lucida (15 cells from control slices and 14 cells from hyperexcitable slices). Local axonal arborizations were more extensive in cells of hyperexcitable slices. This increase in axon collaterals was generally seen in the alveus and in stratum oriens, but changes were more prominent in the latter. In stratum oriens, cells from hyperexcitable slices showed a significant increase in mean total axon length (1035 versus 373 μm in control), in mean number of branching points (6.50 versus 0.67 in control) and in mean number of segment orders per axon (3.07 versus 1.47 in control). Their first‐order axon segments were similar in length to those of control cells (236 versus 338 pm in control), but with significantly more branching points (2.86 versus 0.53 in control). Their second‐order axon segments were significantly longer (381 versus 63 μm in control) and also showed more branching points (2.71 versus 0.13 in control). Their third‐ and fourth‐order axon segments were also longer and with more branching points. Under high‐power light microscopic examination, biocytin‐labelled axonal varicosities in cells of hyperexcitable slices were often seen in close apposition with their own dendrites, presumably making synaptic contact (five of nine cells examined). No such appositions were seen in any of the control cells (seven cells examined). These results indicate that, following kainate lesions, there is sprouting of local axon collaterals of CA1 pyramidal cells in stratum oriens and in the alveus. This local increase in axon collaterals may contribute to the epileptiform activity in the CA1 area by providing recurrent excitation via newly formed synaptic, and perhaps even autaptic, contacts with pyramidal cell dendrites.

Selective loss of GABA neurons in area CA1 of the rat hippocampus after intraventricular kainate

The intraventricular injection of kainic acid (KA) in rats produces a loss of dentate hilar neurons and hippocampal CA3 pyramidal cells, and renders the dentate granule cells and the CA1 pyramidal cells hyperexcitable. We have used immunocytochemical detection of glutamic acid decarboxylase (GAD), a marker of gamma-aminobutyric acid (GABA) cells, as well as stereological cell counting techniques, to determine whether inhibitory cell loss was present 2 weeks after KA treatment. In area CA1, we found that the density of GAD-positive cells was reduced by KA, but only in stratum oriens and the alveus. Counts of Nissl-stained neurons were also significantly reduced in this layer. These results demonstrate a loss of GABA cells in the basal dendritic layer of the CA1 region, which may underlie the hyperexcitability of CA1 pyramidal cells following KA treatment.

beta-Actin is confined to structures having high capacity of remodelling in developing and adult rat cerebellum.

Neurons undergo complex morphological changes during differentiation and in cases of plasticity. A major determinant of cell morphology is the actin cytoskeleton, which in neurons is comprised of two actin isoforms, non‐muscle γ‐ and β‐actin. To better understand their respective roles during differentiation and plasticity, their cellular and subcellular localization was examined in developing and adult cerebellar cortex. It was observed that γ‐actin is expressed at a constant level throughout development, while the level of β‐actin expression rapidly decreases with age. At the light microscopic level, γ‐actin staining is ubiquitous and the only developmental change observed is a relative reduction of its concentration in cell bodies and white matter. In contrast, β‐actin staining almost completely disappears from the cytoplasm of cell bodies, primary dendrites and axons. In young cerebellar cultures, γ‐actin is found in the cell body, neurites and growth cones, while β‐actin is mainly found in growth cones, as previously reported in other primary neuronal culture systems [Kaech et al. (1997), J. Neuroscience, 17, 9565–9572; Bassell et al. (1998), J. Neuroscience, 18, 251–265]. Electron microscopy of post‐embedding immunogold‐labelled tissue confirms the widespread distribution of γ‐actin, and also reveals an increased concentration of γ‐actin in dendritic spines in the adult. During development, β‐actin accumulation is observed in actively growing structures, e.g. growth cones, filopodia, cell bodies and axonal tracts. In the adult cerebellar cortex, β‐actin is preferentially found in dendritic spines, structures which are known to retain their capacity for morphological modifications in the adult brain. This differential subcellular localization and developmental regulation of the two actin isoforms point to their different roles in neurons.

QUANTIFIED DISTRIBUTION OF SEROTONIN TRANSPORTER AND RECEPTORS DURING THE POSTNATAL DEVELOPMENT OF THE RAT BARREL FIELD CORTEX

Serotonin membrane transporter and 5-HT1B and 5-HT2A receptors were visualized and measured by autoradiography in the rat barrel field cortex at postnatal days 4, 8, 12, 16 and in adult (> P60). [3H]citalopram binding, reflecting the presence of 5-HT transporter on thalamocortical fibers, produced a clearcut barrel pattern from P4 to P16 (peak at P8), and decreased to a dispersed, low density in the adult. The patterning and temporal profile of 5-HT1B receptor binding ([125I]cyanopindolol) followed a parallel course. The 5-HT2A receptor binding ([125I]DOI) also conformed transiently to a barrel pattern; it increased in density from P8 to P16 and returned to a level as low as at P4 in the adult. These data suggest that 5-HT exerts a dual role in the developing somatosensory cortex: a local regulation of the peripherally-induced activity of thalamocortical axons via 5-HT1B receptors, and a trophic-like influence mediated by 5-HT2A receptors and possibly involving BDNF.

Increased number and size of dendritic spines in ipsilateral barrel field cortex following unilateral whisker trimming in postnatal rat.

The barrel field area of the primary somatosensory cortex of rodents is a fertile ground for investigating experience‐dependent plasticity and its mechanisms, because the neurons in its layer IV are distributed in groups (barrels) which correspond somatotopically to the vibrissae of the contralateral facial pad. After removal of three rows of whiskers from the right facial pad of young rats during the first two postnatal months, we looked for eventual changes in dendritic spine number and morphology in the corresponding barrels ipsi‐ and contralateral to the deprivation. Intact littermate controls were also examined. Spine number was determined by means of the unbiased disector method in electron micrographs from serial thin sections processed for post‐embedding gamma‐aminobutyric acid (GABA) immunocytochemistry. The volume and surface area of spine head, surface area of postsynaptic density and length of spine neck were measured from computerized three‐dimensional reconstructions. Even though there was no significant side‐to‐side difference in the numerical density of dendritic spines in the experimental animals, the total number of spines in the ipsilateral barrels had increased by 67%, in view of the greater thickness of layer IV on this side. Moreover, spine head volume and surface area of postsynaptic densities were increased, and the length of spine neck was reduced in the ipsilateral compared to the contralateral cortex, and similar differences were noticeable between ipsilateral and control cortex. These changes apparently involved not only the predominant population of relatively small, dendritic spines innervated by asymmetrical synaptic terminals, but also the relatively small contingent of larger spines receiving symmetrical synapses formed by GABA terminals. The most likely explanation for such ipsilateral changes was an increased use of the intact (contralateral) facial pad during postnatal life, in keeping with the notion that activation of a peripheral sensory apparatus during the early postnatal period may have profound effects on the neuronal morphology and structural design of the primary somatosensory cortex. A possible mechanism in this case might be the excessive early activation of thalamic afferents, resulting in increased production of trophic factors, such as brain‐derived nerve growth factor. J. Comp. Neurol. 400:110–124, 1998.

Alterations of perisomatic GABA synapses on hippocampal CA1 inhibitory interneurons and pyramidal cells in the kainate model of epilepsy

In the kainate model of epilepsy, electrophysiological and anatomical modifications occur in inhibitory circuits of the CA1 region of the rat hippocampus. Using postembedding GABA immunocytochemistry and electron microscopy, we characterized perisomatic GABA and non-GABA synaptic contacts in CA pyramidal cells, and GABAergic interneurons of stratum oriens/alveus and stratum lacunosum-moleculare, and examined if changes occurred at these synapses at two weeks post-kainate treatment. We found that, in control rats, the number and total length of perisomatic GABA synapses were significantly smaller (approximately 40-50%) in lacunosum-moleculare interneurons than in oriens/alveus interneurons and pyramidal cells. Additionally, the number and total length of perisomatic non-GABA synapses were different among all cell types, with these parameters increasing significantly in the following order: pyramidal cells<lacunosum-moleculare interneurons<oriens/alveus interneurons. Following kainate treatment, we found that the number and total length of GABA synapses were significantly increased in lacunosum-moleculare interneurons (by 76% and 100%, respectively), but were unchanged in pyramidal cells and oriens/alveus interneurons. In addition, the mean length of individual GABA synapses was significantly increased (by 17%) in pyramidal cells after kainate treatment. In contrast, no changes were observed at non-GABA synapses in any cell type examined after kainate treatment. These results indicate that, in control animals, the ultrastructural correlates of perisomatic GABA inhibition are less pronounced in lacunosum-moleculare than oriens/alveus interneurons or pyramidal cells, whereas those of perisomatic excitation are more prominent in oriens/alveus than lacunosum-moleculare interneurons, and much less present in pyramidal cells. In addition, our results with kainate-treated animals suggest that cell-specific changes in perisomatic inhibition may occur in CA1 inhibitory interneurons in the chronically hyperexcitable hippocampus. The ultrastructural correlates of perisomatic inhibition were increased in lacunosum-moleculare interneurons, which may thus suggest some disinhibition of pyramidal cells. However, the ultrastructural correlates of perisomatic inhibition were increased in pyramidal cells, implying some enhancement of perisomatic inhibition of principal cells in the hyperexcitable hippocampus.

Tau-mediated process outgrowth is differentially altered by the expression of MAP2b and MAP2c in Sf9 cells †

It is well documented that the MAPs, MAP2 and tau, play pivotal roles in neurite outgrowth. Several isoforms of MAP2 and tau are coexpressed in neurons, suggesting that the pattern of neurite outgrowth results from a functional equilibrium among these isoforms. In the present study, by coexpressing two of these MAPs at the same time in Sf9 cells, we demonstrated that tau-mediated process outgrowth is affected differently by MAP2b and MAP2c. MAP2b impairs tau ability to induce process outgrowth. Tau affects MAP2c capacity to induce the formation of multiple processes. There is evidence that actin microfilaments (F-actin) are involved in the elaboration of tau-mediated process outgrowth in Sf9 cells. We compared the effects of MAP2b and MAP2c with the effects of tau on F-actin distribution and stability in Sf9 cells. In MAP2b- and MAP2c-expressing cells with processes, F-actin was redistributed. However, in MAP2b-expressing cells without processes, the distribution of F-actin appears to be similar to the one in wild-type infected cells. Collectively, these results indicate that MAP2b could impair the ability of MAP2c and tau to redistribute F-actin in Sf9 cells, thereby decreasing their capacity to induce process formation. Furthermore, MAP2b and MAP2c patterns of process outgrowth were differentially modified by depolymerization of F-actin by cytochalasin D (CD). As previously reported for tau-expressing cells, the MAP2b-expressing cells developed a higher number of processes per cell and a higher number of cells presented processes in the presence of CD. However, the number of cells with multiple processes was lower in MAP2b-expressing cells than in tau-expressing cells treated with CD at 24 h postinfection. This suggests that MAP2b exerts an effect on F-actin stability at an earlier stage of infection than tau. MAP2c had also some stabilizing effects on F-actin at an early stage of infection, since the percentage of cells presenting one process was similar to the nontreated cells. Therefore, MAP2b seems to have less capacity than MAP2c to redistribute F-actin but, nonetheless, both of these MAP2 isoforms exert a stabilizing effect on F-actin at an early stage of infection. Finally, by modifying phosphorylation we showed that MAP2c capacity to induce multiple processes is related to protein phosphorylation in Sf9 cells. Therefore, the differential effect of MAP2c and MAP2b on process outgrowth seems also to depend on protein phosphorylation.

Developmental changes in expression of myotonic dystrophy protein kinase in the rat central nervous system

Myotonic dystrophy protein kinase (DMPK) is the protein product of the genetic locus associated with myotonic dystrophy, in which alterations of muscle excitability, cardiac conduction defects, mental retardation, and cognitive deficiencies are inherited as an autosomal dominant trait. DMPK belongs to a novel protein serine/threonine kinase family, but its regulation and physiological functions have not been specified. In a first step toward understanding the functions of DMPK in the central nervous system, we have characterized its localization and developmental pattern of expression in rat brain and spinal cord by using a monospecific rabbit antiserum produced against bacterially expressed DMPK. Expression of DMPK begins after birth and increases gradually to peak at postnatal day 21 with antibody labeling of neuronal cell types in many regions. After postnatal day 21 and proceeding to the adult, the pattern of expression becomes more restricted, with localization to certain regions or cell groups in the central nervous system. Electron microscopy reveals localization within adult spinal motor neurons to the endoplasmic reticulum and dendritic microtubules. The adult localizations suggest that DMPK may function in membrane trafficking and secretion within neurons associated with cognition, memory, and motor control. J. Comp. Neurol. 394:309–325, 1998.

Equivalent cell density in three areas of neonatal rat cerebral cortex.

Using the disector method, equivalent cell densities (number of cells per unit volume) were found in the frontal (motor), parietal (somatosensory) and occipital (visual) cortex of neonatal rat (1.2 x 10(6) cells/mm3). As determined with GABA post-embedding immunocytochemistry on semithin sections, at least 11% of these cells expressed GABA. Because neonate frontal cortex is thicker than the parietal or the occipital cortex, the number of cells under an unit area of pial surface was greater in this cortex. This clearly differed from the adult pattern, where the greatest number of cells beneath an unit pial area is that of the somatosensory versus the frontal and the occipital cortex. It suggests that the attainment of the adult number of cells in a cortical column is achieved through differential cell death in the different areas and that the amount of thalamic input could be a crucial determinant of the adult cytoarchitecture.