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. 2004 Mar 24;24(12):2853-65.
doi: 10.1523/JNEUROSCI.4814-03.2004.

Axon branching and synaptic bouton phenotypes in GABAergic nonpyramidal cell subtypes

Fuyuki Karube  1 , Yoshiyuki Kubota, Yasuo Kawaguchi
Affiliations

Axon branching and synaptic bouton phenotypes in GABAergic nonpyramidal cell subtypes

Fuyuki Karube et al. J Neurosci. .

Abstract

GABAergic nonpyramidal cells, cortical interneurons, consist of heterogeneous subtypes differing in their axonal field and target selectivity. It remains to be investigated how the diverse innervation patterns are generated and how these spatially complicated, but synaptically specific wirings are achieved. Here, we asked whether a particular cell type obeys a specific branching and bouton arrangement principle or differs from others only in average morphometric values of the morphological template common to nonpyramidal cells. For this purpose, we subclassified nonpyramidal cells within each physiological class by quantitative parameters of somata, dendrites, and axons and characterized axon branching and bouton distribution patterns quantitatively. Each subtype showed a characteristic set of vertical and horizontal bouton spreads around the somata. Each parameter, such as branching angles, internode or interbouton intervals, followed its own characteristic distribution pattern irrespective of subtypes, suggesting that nonpyramidal cells have the common mechanism for formation of the axon branching pattern and bouton arrangement. Fitting of internode and interbouton interval distributions to the exponential indicated their apparent random occurrence. Decay constants of the fitted exponentials varied among nonpyramidal cells, but each subtype expressed a particular set of interbouton and internode interval averages. The distinctive combination of innervation field shape and local axon phenotypes suggests a marked functional difference in the laminar and columnar integration properties of different GABAergic subtypes, as well as the subtype-specific density of inhibited targets.

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Figures

Figure 5.
Figure 5.
A, Two kinds of branching angles at an axon node, aperture, and tilt angle. A1, Distribution of aperture angles of a single cell (neurogliaform cell). Thick line, Fitted Gaussian. A2, Distribution of tilt angles of a single cell (neurogliaform cell). Thick line, Fitted gamma distribution. The shape parameter of the gamma is 2.12. B, Distribution of vertical direction change at a node of a single cell (Martinotti cell). Thick line, Fitted Gaussian distribution. C, Distributions of means and SDs of aperture angles, tilt angles, and vertical direction changes of all cells. D, Reconstruction of a Martinotti cell. The soma and dendrites are black, and the axons are gray. Top, Note the characteristic ascending axonal arborizations. Bottom, Enlargement of the soma and axon initial portion. Note that the axons emerged from the white matter side of the soma but changed the direction and generated ascending collaterals.
Figure 7.
Figure 7.
A1, A2, Autocorrelograms of bouton arrangements along the axons of Martinotti (1401 boutons) and FS basket cells (1878 boutons). Right, Enlargements to 12 μm. Thick line, Fitted sigmoid function; dotted line, half value of sigmoid function. Bin, 0.5 μm. B, The way to construct one-dimensional bouton lines from the nested axon dendrogram for calculating the autocorrelogram. C, The cumulative histogram of the bouton length along the axon of 230 boutons reconstructed from serial ultra-thin sections of seven nonpyramidal cells. Thick line, Fitted sigmoid function; dotted line, half value of sigmoid function. Bin, 0.1 μm. D, Three types of axonal boutons different in junction number and intrabouton location. D1, Boutons with a single junction area. Two successive boutons were reconstructed. D2, A bouton with two junction areas on the same side. D3, A bouton with two junction areas on the opposite sides. Two views of the bouton with 180° rotation are shown. E, Intervals between synaptic junction centers in three types of successive junction relationships. F, Schematic view of the synaptic bouton arrangement of nonpyramidal cells. G, Frequency distribution of the intervals of two successive boutons on the same somata, obtained from two large basket cells. H, The autocorrelogram of bouton arrangements along the axons of an FS chandelier cell (2049 boutons).
Figure 1.
Figure 1.
A–C, Firing responses of nonpyramidal cells induced by depolarizing currents. r.p., Resting potential. Membrane potentials changed by DC are indicated in parentheses. A1, A2, FS cells. B, A LS cell. C1–C4, Non-FS cells. D1, Distributions of widths of intervaricose axons and synaptic boutons of an FS basket cell. D2, Reconstruction of axon terminals (marked by 1 and 2) and the postsynaptic dendrite (pale). The axon terminal 1 from the FS cell made a symmetrical synapse on the shaft. The synaptic junctional areas are shown on the right side. D3, Two views of the FS cell synaptic bouton at right angles. E, Boutons of an FS basket cell and their appositions on unstained somata (1, 2) observed with differential interference contrast. F, Schematic representation of the axon collaterals and boutons shown in E. Identified postsynaptic targets: s, soma; d, dendritic shaft; sp, spine; d+sp, a bouton making two synapses on a dendritic shaft and a spine; v, synaptic vesicles were observed, but the junction could not be identified. G, H, Synaptic contact of the bouton apposition on the soma (E, b1). s, Soma. I, Dendritic synaptic contact of the identified bouton (E, b2). d, Dendrite. J, Distribution of the somatic bouton percentage of nonpyramidal cells. Nonpyramidal cells were divided into cells with a low and high proportion of somatic boutons.
Figure 2.
Figure 2.
Nonpyramidal cell subtypes. A, FS cell subtypes. a, FS basket cell in layer V. b, FS chandelier cell in layer II/III. Roman numerals correspond to the cortical layers. The somata and dendrites are shown in black, and the axons in red. c, Distribution of the axonal node frequency and maximum bouton density. soma bt, Somatic bouton percentage. B, LS cell subtypes. a, LS neurogliaform cell in layer II/III. b, LS basket cell in layer II/III. c, The number of primary dendrite and the dendritic node frequency. C, Non-FS cell subtypes. The identified peptides are shown in parentheses. a, Large basket cell. b, Small basket cell. c, Descending basket cell. d, Distribution of the axonal node frequency and somatic volume of non-FS basket cells. e, Martinotti cell. f, Double bouquet cell. g, The axonal node frequency and proportion of boutons on the white matter side of Martinotti and double bouquet cells.
Figure 9.
Figure 9.
A, Relationships of the internode interval (mean) and the interbouton interval (median) of nonpyramidal cell subtypes. A1, FS basket, FS chandelier, and LS neurogliaform cells. *, neurogliaform cell with sparse innervation. A2, Martinotti and double bouquet cells. A3, Small, descending, large, and LS basket cells. Each subtype showed a distinct combination of the mean internode intervals and the median of interbouton intervals. B, Significant differences between pairs of nonpyramidal cell subtypes in the mean internode and median interbouton intervals (α < 0.05; Tukey-HSD test). C, Schematic view of the axon internode and interbouton intervals along the axon of nonpyramidal cells. D, Combination of parameter values for the bouton and branching phenotypes (interbouton interval median and internode interval mean) and those for the bouton spatial distribution (bouton horizontal and vertical spread around soma) in four subtypes of nonpyramidal cells. For the interlayer comparison, both layer II/III and V cells are shown for FS basket, Martinotti, and double bouquet cells. Note that a characteristic association of four parameters in each subtype and the similarity between layer II/III and V cells.
Figure 3.
Figure 3.
A, Cluster analysis of non-FS cells using the somatic bouton percentage, somatic volume, axonal node frequency, and proportion of boutons on the white matter side. Ordinate, individual cells labeled with positive immunohistochemical markers; abscissa, the average within-cluster linkage distance. B, Relationship of layer II/III GABA cell subpopulations positive for CCK, calretinin (CR), CRF, somatostatin (SOM), or VIP. The relative number of cells immunoreactive for a substance is proportional to the size of the box. C, The vertical and horizontal bouton spread around soma in nonpyramidal cell subtypes. C1, C2, Basket cell subtypes and the others, respectively. Cells marked by the asterisk were in layer V, and the others in layer II/III. D, Statistical comparison of the bouton spatial distribution among nine nonpyramidal cell subtypes, using an ANOVA/post hoc test (Tukey-HSD). Cell types are ranked in descending order of the mean for individual parameters. D1, Vertical spreads. D2, Horizontal spreads.
Figure 4.
Figure 4.
A, Linear relationship of internode direct distances and intervals of a single cell (large basket cell). Solid line, Simple regression fit. Tortuosity, ratio of the internode interval (L) to the direct distance (D). B, Axon internode interval. a, b, Frequency distributions of internode intervals of two cells with shorter and longer means (small and large basket cells, respectively). Thick line, Fitted exponential distribution; λ, decay constant of the fitted exponential (x, interval). c, Linear relationship of the SD and mean of internode intervals of all cells. a and b correspond to cells shown in a and b, respectively. Solid line, Simple regression fit; c.c., correlation coefficient; r.c., regression coefficient. d, Linear relationship of the decay constant (λ) and the mean of internode intervals.
Figure 8.
Figure 8.
Statistical comparison of the axon local characteristics among nine nonpyramidal cell subtypes, using an ANOVA/post hoc test (Tukey-HSD). Cell types are ranked in descending order of the mean for individual parameters. A, Means of tortuosity. B, Means of the internode interval. C, Means of aperture angle. D, Means of tilt angle. E, Medians of interbouton interval.
Figure 6.
Figure 6.
Synaptic bouton distributions along the axon. A, Linear relationship of the bouton number and internode interval of a single cell (Martinotti cell). Solid line, Simple regression fit; c.c., correlation coefficient. B, Ratio of boutons in the far region to those in the near near one, suggesting the homogeneous distribution along the axon independent of the distance from the soma. Near region, The sphere containing one-third of the total axon length; far region, outside the sphere containing the two-thirds. C, Axon interbouton interval. a, b, Frequency histograms of the interbouton intervals of two cells with shorter and longer medians (Martinotti and FS basket cells, respectively). Thick line, Fitted exponential distribution from the peak. c, Linear relationship of the SD and mean of interbouton intervals. a and b correspond to cells shown in a and b, respectively. Solid line, Simple regression fit; r.c., regression coefficient. d, Linear relationship of the decay constant (λ) and the median of interbouton intervals.
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