Synaptic organization of GABAergic neurons in ... - Wiley Online Library

THE JOURNAL OF COMPARATIVE NEUROLOGY 2621-12 (1987)

Synaptic Organization of GABAergic Neurons in the Mouse SmI Cortex ASAF KELLER AND EDWARD L. WHITE Department of Morphology, Corob Center for Health Sciences, Ben-Gurion University of the Negev, Beer Sheva, Israel; and Institute for Neurologic Research, Inc., Arlington, Massachusetts

ABSTRACT Immunocytochemical methods were used to examine GABAergic neurons in the barrel region of the mouse primary somatosensory cortex. GABAergic neurons occur in all layers of the barrel cortex but are more concentrated in the upper portion of layers IHII and in layers IV and VI. Nine cells in layer IV were examined with the electron microscope, and portions of their dendrites were reconstructed from serial thin sections. These cells are of the nonspiny, multipolar or bitufted varieties, and some of them have beaded dendrites. The labeled cell bodies and their reconstructed dendrites were postsynaptic at asymmetrical synapses with thalamocortical axon terminals labeled by lesion-induced degeneration and with unlabeled axon terminals. Each cell also received symmetrical synapses from GABAergic axon terminals and from unlabeled axon terminals. Our results indicate that GABAergic cell bodies and processes receive synapses from thalamocortical axon terminals but that different cells display marked differences in the proportion of thalamocortical and other synapses they receive. These results indicate that GABAergic cells form a heterogeneous population with respect to their morphologies and patterns of synaptic inputs. The synaptic sequences revealed here for GABAergic neurons represent a n anatomical substrate for various inhibitory processes known to occur within the cerebral cortex. Key words: GABA, GAD,intrinsic circuitry, cerebral cortex, cortical inhibition

Gamma-aminobutyricacid (GABA)has been identified as the major inhibitory transmitter utilized by cortical neurons in several mammalian species (Krnjevic and Schwartz, '67; Dichter, '80; Krnjevic, '841, and GABA-mediated inhibition has been demonstrated to play a vital role in determining the functional characteristics of cortical neurons (e.g., Tsumoto et al., '79;Dykes et al., '84; Sillito, '84). Recent light microscopic studies have used immunocytochemical methods to identify GABAergic neurons in layer J Y of the region of the mouse primary somatosensory cortex (Lin et al., '85; Chmielowska et al., '86; Keller and White, '86) that is related to the large mystacial vibrissae on the animal's snout. Layer IV of this region of cortex was termed the posteromedial barrel subfield (PMBSF) by Woolsey and Van der Loos ('70). A large body of information has been provided on the synaptic organization of cells in the PMBSF cortex in mice, particularly concerning thalamocortical input to this region (see reviews by White, '79, '81, '86). Yet there is no information regarding thalamocortical and other synaptic connections of GABAergic cells in this or for that matter in any other region of the cortex. The aim of the

0 1987 ALAN R. LISS, INC.

present study is to rectify this deficiency by examining thalamocortical and other synapses with labeled GABAergic neurons in the mouse PMBSF cortex. Our aim was to determine whether GABAergic cells form a homogeneous functional group with respect to their synaptic input and to attempt to incorporate the synaptology of these cells in the functional organization ?f the mouse SmI cortex.

MATERIALS AND METHODS Two- to 3-month old male CD/1 mice were used in this study. The animals were anaesthetized and electrolytic lesions were placed in the nuclei ventralis posterior pars lateralis (Sidman et al., '71; VBm nucleus of Van der Loos, '76). A posterior approach through the tectum was used so as not to damage other regions of the brain that project to or from the PMBSF cortex (White and DeAmicis, '77). Four days after the lesions, the mice were anaesthetized and perfused intracardially with a fixative containing 4% paraAccepted January 21,1987.

2

A. KELLER AND E.L. WHITE

formaldehyde, 0.05%glutaraldehyde, and 0.2% picric acid (Somogyi and Takagi, '82). The brains were removed, sectioned at 40 pm with a vibratome (Lancer, U.S.A.), and processed for immunocytochemistry according to the avidin-biotin horseradish peroxidase method (ABC; Bio-Yeda, Israel) of Hsu et al. ('81). Sections were processed with either of the following antisera: anti-GABA (Immunonuclear, Minnesota), diluted 1:3,600, or anti-GAD (developed and characterized Drs. I.J. Kopin, W. Oertel, D.E. Schmechel, and M. Tappaz of the Laboratory of Clinical Science, NIMH), diluted 1:3,600. Control sections were processed with a preimmune serum diluted 1:1,000. Sections containing the thalamus were examined with the light microscope to assess the location and extent of the lesion. In each case, the lesion involved the entire nucleus ventralis thalami, pars lateralis, as well as the nucleus posterior thalami, which also projects to layer IV of the barrel cortex in mice (White and DeAmicis, '77). Parts of other thalamic nuclei were occasionally lesioned, but none of them is known to project to the mouse somatosensory cortex. The location of PMBSF cortex and of the laminar boundaries within it were determined by superimposing onto the vibratome sections drawings made from a set of Nisslstained coronal sections of comparable thicknesses in which the barrels and laminar boundaries are clearly visible. Nine cells in layer IV of the PMBSF cortex that were immunoreactive for either GAD or GABA were processed for electron microscopy. Unbroken series of silver-gold sections (90 nm thick, Williams and Meek, '66) were sectioned, and examined with a JEOL lOOSX electron microscope. Somata and processes of the GABAergic cell bodies were examined on each thin section of the series to determine their size and the numbers and types of synapses they received. Labeled dendrites, belonging to the GABAergic cells, were traced from their somata for long distances and micrographed on every thin section to a magnification of X 7,600; 70-mrn roll film was used. Strips of negatives, each containing all the micrographs taken of a single thin section through a labeled dendrite, were placed in succession on a light box and examined with a ~ 1 lens. 0 All the synapses observed on a labeled profile were identified, and their locations were marked on the negatives. Distances between the synapses were measured directly from the negatives when the labeled profiles were sectioned longitudinally. In instances in which the long axis of the profiles was a t an angle oblique to the plane of section, trigonometry was used to calculate distances along the processes. By using relatively constant structures within the sections, such as large cross-sectioned processes, successive sections were placed in register and the outlines of the labeled profiles and their synapses were traced onto a single sheet of paper. The sequential profiles, as they appeared in successive negatives, were displaced one from the next by a distance of 0.7 mm, which corresponded to the thickness of the sections multiplied by the magnification of the negative.

RESULTS Light microscopy GABA- and GAD-positive reaction product was observed in neuronal cell bodies and in their proximal processes, in puncta, which were presumably axon terminals and in cross sections through other, presumed, neuronal processes. No reaction product was observed in control sections. Cell bodies were found in all layers of the barrel cortex but were

more concentrated in the upper portion of layers IMII and in layers IV and VI. Cell bodies in layer IV were spherical, with processes radiating in all directions, or vertically oriented and elongate, emitting processes only from their poles. None of the labeled cells possessed an apical dendrite typical of cortical pyramidal cells. Only the proximal dendrites were seen with the light microscope, and these were devoid of spines. Occasionally, a short tapering process, resembling an initial axon segment, originated from the soma or from a primary dendrite. Puncta were distributed throughout the cortical layers, with the highest density characterising layer IV and a somewhat lower concentration in layer VI. These findings are consistent with a previous light microscopic report of the shape and laminar distribution of GABAergic neurons in the mouse PMBSF cortex (Keller and White, '86).

Electron microscopy In thin sections, GAD-positive reaction product appeared as a fine, particulate, electron-dense material (Fig. 11, staining the cytoplasm of neuronal cell bodies, dendrites, axons, and axon terminals. GAD-positive reaction product was most prominent along the cisternae of the Golgi aperatus and rough endoplasmatic reticulum and on the surfaces of mitochondria and synaptic vesicles. GABA-positive reaction product was less particulate and more diffuse than the one produced by incubation with GAD (Fig. 2); GABApositive reaction product stained the nucleus as well as the cytoplasm and processes. No other qualitative or quantitative differences were observed between GAD- and GABApositive cells; hence GABA-positive and GAD-positive neurons will be referred to collectively as GABAergic neurons.

Layer IV GABAergic neurons Neurons were classified according to the criteria of Peters and Regidor ('81):Multipolar cells were those having dendrites that radiated in all directions from the cell body, whereas bitufted cells had tufts of dendrites that extended preferentially from two opposite poles of the soma. A few GABAergic cells, which appeared as bipolar cells with the light microscope, proved to be of the bitufted variety when the full complement of their dendrites was revealed in serial thin sections. True bipolar cells, having single dendrites originating from opposite poles of the soma, were not encountered in our preparations. The types and sizes of the GABAergic neurons studied are presented in Table 1, in which data are presented, from top to bottom, according to increasing size of the cell body. Of the nine cells examined in this study, six cells were classified as multipolar and the others were bitufted. The size of each of the GABAergic cell bodies was measured directly from the electron micrographs. Cell bodies ranged in size from 8.2 x 11.3 pm for the smallest cell to 17.9 x 20.6 p m for the largest. Two of the bitufted cells (cells A and B) possessed the smallest somata of the labeled cells, whereas the third bitufted cell (cell G) had a soma of medium size. The multipolar cells tended to have larger somata than those of bitufted cells. Barrels cannot be visualized directly in thin sections through the PMBSF, but the location of the septa between barrels can be inferred by the presence of numerous myelinated and unmyelinated axons and by the paucity in septa of cell bodies and degenerating thalamocortical axon terminals (White, '76, '78). In addition, as pointed out by Feldman and Peters ('74) in their study of rat barrel cortex, barrel walls are characterized by strings of closely spaced

3

GABAERGICNEURONS

Figs. 1, 2. Thin sections through GABAergic cell bodies and proximal dendrites of multipolar neurons in layer IV reacted with an antibody to GAD (Fig. 1) or to GABA (Fig. 2). Note the difference in the reaction product produced by incubation with the different antisera and the fact that anti-GAD stains only the cytoplasm, whereas anti-GABA stains the nucleus as well. An unlabeled cell body is marked with the asterisk in Figure 1. Figure 1, ~7,000; Figure 2, ~4,000.

cell bodies. These criteria were used to determine whether GABAergic cell bodies were located in barrel walls, hollows, or septa. None of the neurons studied had a cell body in a barrel septum. Only two of the somata studied, belonging to cells F and I, both identified as multipolar, were situated in barrel hollows; six other cells were clearly situated near barrel walls, whereas the location of cell E was difficult t o define precisely.

The cytoplasm of GABAergic cells was best studied in preparations that were produced with GAD as the antiserum, because the reaction product does not obscure ultrastructural details and the nucleus remains unstained. The nucleus showed no unique nuclear inclusions and usually possessed more than one nucleolus. A characteristic feature of the cytoplasm in GABAergic cell bodies was the very large number of mitochondria (Fig. 1).


4 TABLE 1. Synapses With Reconstructed GABAergic Neurons' Synapses Cell bodies

Cell type

S

A

S/A

GABA(%)

TC(%)

11.3

2

17

0.1

2(100)

3(17.6)

Diameter(pm)

8.2

Dendrites Length No. pm* A1 A2 B

A

bituf

B C

bituf. multi.

9.7 x 12.6 10.5 x 11.2

5 7

24 14

0.2 0.5

4(80) 6 (85.7)

11 (45.8) g(64.3)

D

multi.

10.1 x 14.4

3

9

0.3

Z(66.7)

3 (33.3)

D1 D2

E

multi. multi.

11.6 x 9.2 12.3 x 13.1

3 11

21 36

0.1 0.3

3(100) g(81.8)

14(66.7) 7 (19.4)

E F1 F2

bituf. multi. multi.

13.2 x 16.6 13.1 x 19.4 17.9 x 20.6

7 17 13

66 160 94

0.1 0.1 0.1

7 (100) 14182.4) 11 (84.6)

46t28.8)

G H

22 (23.4)

I1

F G

H I

X

6(9.1)

C1 C2

I2

53.7 (0.4) 23.9(1.0) 38.6U.7) 53.0(0.2) 24.9(1.0) 54.7 (0.8) 75.5 (0.9) 64.7 (0.6) 33.8(0.8) 73.9(0.6) 90.1 (1.3) 61.5 (1.01 92.6(1.0) 36.7 (0.8)

Synapses S

A

S/A

GABA(%)

TC(%)

3 2

19 22 62 10 24 42 58 36 22 41 106 56 90 27

0.2 0.1 0.05 0.1

3(100)

3(15.8) 3U3.6) 2 (3.2) l(10.0) 3(12.5) 2(4.8) 3 (5.2) 3(8.3) 3113.6) 8(19.5) a(7.5) B(14.2) S(3.3) l(3.7)

3 1 2

4 9 4 4 4 8

5 7 3

0.08 0.1 0.1 0.1 0.2 0.1 0.08 0.09

0.08 0.1

2(100)

3 (100) l(100) 2 (100) 3(75) 6(66.7) 3(75) 3(75) 3(75) 6(75) 5(100) 5 (71.4) 3(100)

~~

'Abbreviations: multi.: multipolar; bituf.:bitufted; SYM:symmetrical, including GABA; ASYM asymmetrical, including TC;S / A ratio of symmetrical to asymmetrical synapaes; GABA: GABAergic terminals; (7%):GABA a8 a percent of SYM; TC:thalamocorticat (a): thalamocortical as a percent of ASYM. 'Figures in parentheses represent average number of synapses per micron length.

Dendrites Fourteen dendrites were traced in serial thin sections from GABAergic cell bodies. These dendrites were labeled with reaction product for varying distances from the cell body but rarely beyond the branch points of secondary dendrites. In mbst cases the dendrites were traced distal to the point where the labeling terminated, and thus it was possible to reconstruct large portions of the dendrites to determine their shapes and the location, types, and number of synapses they received, Reconstructed dendrites belonging to five of the GABAergic neurons examined are represented in Figure 3, which shows the location of thalamocortical synapses with their shafts. All of the reconstructed dendrites were essentially smooth or nonspiny, although one cell (D) possessed two spines on one of its dendrites and a third spine on a second dendrite (Fig. 3). These spines received asymmetrical synapses from unlabeled terminals. Most of the dendrites reconstructed featured occasional varicosities along their shafts, giving the dendrites a beaded appearance, but because of the limitations of two-dimensionalgraphics it is not always possible to appreciate this shape from the reconstructions (Fig. 4). Most of the reconstructed dendrites were oriented vertically, projecting either toward the pial surface or toward the white matter. Dendrites D1 and G were oriented horizontally and projected outside of the barrel boundaries, each entering an adjacent barrel. Previously, Golgi-impregnated neurons in the rat SmI cortex, whose cell bodies occur in or near barrel walls, have been observed to possess dendrites projecting to adjacent barrels (Simons and Woolsey, '84).

axons entered a myelin sheath, traveled for a short distance in myelin, came out of the myelin, and then reentered it after some distance. Myelinated portions of GABAergic axons were devoid of reaction product, but this reappeared at internodes and within their terminals.

Synaptology Synapses were identified as asymmetric or symmetric according to the criteria usually accepted for the identification of chemical synapses (Peters et al., '76).The numbers and types of synapses formed with cell bodies of the nine GABAergic cells and with 14 of their dendrites are presented in Table 1. GABAergic cell bodies and dendrites were postsynaptic at asymmetrical synapses to unlabeled axon terminals (Figs. 7-9) and to degenerating thalamocortical axon terminals (Fig. 10).They received symmetrical synapses from unlabeled and from GAEJAergic terminals (Figs. 5, 9). The total number of synapses identified on the surfaces of the reconstructed cell bodies ranges from 12 synapses for cell D, to 177 for cell H. The number of synapses received by the larger cell bodies exceeded the number received by the smaller somata. However, no relationship was established between the type of the cell and the total number of synapses formed with its cell body. Thus, the cell bodies of the large bitufted (cell G) and large multipolar cells (e.g., cells H and I) each received many synapses, Likewise, the smaller bitufted (cells A and B) and smaller multipolar cell bodies received similar numbers of synapses. There was no correlation between the size or morphology of the soma and the ratio of synaptic types it received.

Axons

Thalamocortical synapses

GABAergic axons originated either from the parent soma or from an ascending primary dendrite a short distance from the cell body. None of the axons examined had axon terminals synapsing with their initial segments. Most of the labeled axons projected initially toward the pial surface, although one neuron (cell B) had an axon that was oriented initially toward the white matter. Some of the axons were unmyelinated, although a few axons entered a myelin sheath 10-15 pm from their origin, after which they travelled for long distances before branching. One of the

Degenerating thalamocortical axon terminals were characterized by a marked increase in the electron density of their axoplasm and a loss of synaptic vesicles (Fig. 10). Thus,the synapses these terminals formed were identified solely on the presence of a synaptic cleft that was wider than the normal extracellular space and by the presence of electron-densematerial adherent to the cytoplasmic surface of the postsynaptic membrane. Degenerating thalamocortical axon terminals formed only asymmetrical synapses. Each GABAergic cell body examined in this study was

6

A. KELLER AM, E L WHITE

postsynaptic at asymmetrical synapses to degenerating thalamocortical axon terminals. There was no correlation between the size or shape of the cell body and the number or proportion of thalamocortical synapses it received. For example, two small bitufted neurons (cells A and B), received 17.6 and 45.8%, respectively, of their somatic asymmetrical synapses from thalamocortical axon terminals, whereas cells E and F, both medium-sized multipolar cells, received 66.7 and 19.4%, respectively, of their somatic asymmetrical synapses from these terminals. Furthermore, GABAergic cells located in barrel hollows (F and I) vs. near barrel walls did not receive a characteristic proportion of thalamocortical synapses either on their somata or on their dendrites. The reconstructed dendrites received various proportions of. thalamocortical synapses on their shafts, and these ranged from 3.2 to 19.5% of their asymmetrical synapses (Table 1).Dendrites belonging to the same GABAergic cell received similar proportions of thalamocortical synapses, whereas dendrites belonging to different cells received different proportions of thalamocortical synapses. The proportion of thalamocortical synapses presynaptic to dendritic shafts was not correlated with the proportion of these synapses presynaptic to the parent soma, Thus, the proportion of thalamocortical synapses received by somata belonging to cells B, C , D, E, €3, and I was two to 15 times greater than the proportion of thalamocortical synapses received by their dendrites, whereas cells A, F, and G received similar proportions of thalamocortical synapses on their somata and dendrites. Samples of small volumes of neuropil, studied in every region in which the GABAergic somata and dendrites occurred, revealed that somewhere between 18 and 22% of the asymmetrical synapses in the neuropil were formed by degenerating axon terminals. This finding indicates that the differences in thalamocortical input to GABAergic somata and dendrites are not due to a corresponding difference i n the proportion of thalamocortical synapses in the surrounding neuropil.

GABAergic synapses GABAergic axons formed en passant and terminal synapses, all of which were of the symmetrical type. In several instances GABAergic axons, followed in serial thin sections, formed three to four synapses with a single cell body, which, on the basis of its shape, was identified as a pyramidal neuron. Postsynaptic targets included GABAergic cell bodies and dendrites (Fig. 5), pyramidal cell bodies (Fig. 6) and their axon initial segments, nonpyramidal GABA (GADhegative cell bodies, and unlabeled dendritic shafts and spines. Similar neuronal elements were observed to be postsynaptic a t symmetrical synapses to GABAergic terminals in the visual cortex of the cat (Freund et al., '83; Somogyi et al., '85) and rat (Ribak, '78) and in the somatosensory and motor cortices of the monkey (Hendry et al., '83; DeFelipe et al., '85). Each of the GABAergic cell bodies and dendrites received symmetrical synapses from GABAergic axon terminals. There was no correlation between soma1 size, shape, or location in the barrel and the numbers or proportions of GABAergic synapses formed with these cells. A small number of unlabeled terminals also formed symmetrical synapses with GABAergic somata and dendrites. In neuropil surrounding the GABAergic cell bodies, 58 unlabeled dendrites were followed for short distances in

serial sections, and of these dendrites 39 were postsynaptic to GABAergic terminals. Nine of these GABAergic synapses were formed with dendritic spines, and the remaining 30 were formed with dendritic shafts. The morphology of these dendrites and the type of cells from which they arose could not be determined.

Spatial distribution of synapses The distribution of the various synaptic types received by ten of the reconstructed dendrites is presented in Figure 4. This figure was constructed by measuring the distances between the synapses among the dendritic shafts from the somatic origin of the dendrites. The locations of all the synapses were plotted along straight lines, each line representing the total length of the dendritic segment reconstructed. The total number of synapses along dendritic shafts ranged from 0.2 to 1.0 synapse per pm (Table 1). The data depicted in Figure 4 indicate that symmetrical synapses, whether they are formed by GABAergic or by unlabeled axons terminals, have a tendency to occur on the proximal portions of dendrites as well as on dendritic branching points. This finding is in agreement with previous reports on the distribution of synapses received by dendrites of nonspiny multipolar cells (e.g., Peters and Fairen, '78; Davis and Sterling, ' 7 9 White and Rock, '81; White et al., '84).

DISCUSSION In our study, antisera for GABA and GAD were used separately to label neuronal cell bodies and processes in layer IV of PMBSF cortex in the mouse. Incubating control sections with a pre-immune serum produced negative results, a finding that demonstrates the specificity of the method. However, the sensitivity of the method cannot be determined from the results, and there is a possibility that not all neuronal elements containing GABA were labeled in our material.

Morphology of GABAergic neurons Because only the primary and at times the secondary dendrites were visible with the light microscope and because only a limited number of dendrites belonging to a single cell were examined with the electron microscope, it was not possible to appreciate the complete dendritic arborizations of the GABAergic cells. Nevertheless, on the basis of the shapes of their somata and reconstructions of some of their dendrites, these cells can be designated unequivocally as nonspiny multipolar or bitufted varieties. This finding is in accord with previous results showing that GABAergic cells in the somatic sensory cortex of the mouse, rat (Lin et al., '85; Keller and White, '86), and monkey (Houser et al., '83), in the motor cortex of the monkey (Houser et al., '831, and in the visual cortex of the rat

Fig. 4. Graphic demonstration of the distribution of synapses onto reconstructed dendrites (thick black lines) of GABAergic neurons. Cell bodies of origin are indicated by black discs whose designations relate to dendrites in Figure 3 and in Table 1. Daughter branches are attached to the parent dendrites at appropriate sites by dashed lines. Synapses with the reconstructed dendrites are depicted at appropriate locations. Short, thin lines projecting upward from the thick lines represent asymmetrical synapses formed with unidentified axon terminals; below the thick lines are shown the locations of symmetrical synapses involving unidentified axon terminals (5) or GABAergic (Gl axon terminals and asymmetrical synapses involving degenerating thalamocortical axon terminals (T).

Vlll

vI

IVI I VI I

0

v /+

IIIV

I Ill I I I I I

II

1

t

d


Fig. 5. Electron micrograph showing a GAD-positive cell body and its proximal dendrite. Clusters of reaction product are marked with arrowheads. The proximal dendrite forms a n asymmetrical synapse with an unlabeled axon terminal (A). Two GABAergic axon terminals (G) are abutting the GAD-positive dendrite, and the inset (arrow) shows one of these terminals forming a symmetrical synapse with dendritic shaft. ~33,000. Inset ~ 4 6 , 0 0 0 . Fig.6. A GABA-positiveaxon terminal (G)forming a symmetrical synapse with an unlabeled cell body belonging to a layer rV pyramid. Note the punctum adhaerens (arrowhead) associated with the synaptic junction. X45,OOO.

(Ribak, '78;Somogyi et al., '84a) and cat (Freund et al., '83; Hamos et al., '83; Somogyi et al., '85) are non- or sparsely spinous nonpyramidal cells. Several attempts have been made to classify GABAergic cells into distinct morphological groups (e.g., Hamos et al., '83; Houser et al., '84). Based mainly on comparisons of light microscopic observations of GABAergic cells and Golgi-impregnated neurons, it has been suggested that GABA neurons in rat cortex may include chandelier cells, neurogliaform cells, and local plexus neurons (Ribak, '78; Houser et al., '84; Somogyi et al., '84b). Furthermore, it has been suggested that GABA neurons in the monkey cortex include, in addition, basket cells and double bouquet cells (Hendry and Jones, '81; Houser et al., '83, '84). Combining Golgi impregnation and immunocytochemistry has enabled certain GABAergic neurons in the visual cortex of the cat to be classified as basket cells (Freund et al., '83; Somogyi

et al., '83). Certain classifications of nonpyramidal neurons are contingent on the ability to visualize the pattern of their axonal arborizations (Peters and Regidor, '81; Fairen et al., '84). Since in our material only the axon initial segment was observed, it was not possible to use the pattern of axonal arborization to assign the GABAergic cells examined in this study to specific morphological subgroups. However, the strikingly different synaptic patterns exhibited by GABAergic neurons, even those whose morphologies are similar, indicate that the GABAergic cells examined in this study consist of a heterogeneous population composed of a large number of functionally different neuronal types. Reconstructions of dendrites belonging to the GABAergic cells was carried out in an attempt to elucidate their threedimensional morphology (Fig. 3). In spite of the limitations of two-dimensional graphics, it can be concluded that these

GABAERGICNEURONS dendrites have smooth-i.e., nonspiny-shafts, with occasional varicosities. Feldman and Peters ('781, in their report on the forms of nonpyramidal neurons in the visual cortex of the rat, note that even dendrites designated as "spinefree" rarely exhibit a total lack of spines; the classification of a cell as nonspiny is based, rather on extremely low numbers of spines along extensive lengths of dendrites. Thus, in this study, the few spines observed on dendrites of one cell are considered to not alter the general conclusion that the dendrites belonging to GABAergic cells are nonspiny. Golgi-impregnated nonspiny neurons similar to the GABAergic cells observed in this study have been reported in layer IV of the somatic sensory cortex of the mouse (Woolsey et al., '75; White et al., '84),rat (Simons and Woolsey, '84), and primate (Jones, '75), in the visual cortex of the rat (Parnavelas et al., '77; Feldman and Peters, '78; Hedlich and Winkelmann, '$21, cat (Lund et al., '79; Peters and Regidor, '811,and primate (Lund, '73;Lund et al., '81), and in the auditory cortex of the rabbit (McMullen and Glaser, '82). With a single exception, each cell in this study possessed an axon that projected initially toward the pial surface. Pertinent to this is the recent finding by DeFelipe and Jones ('85) that GABA-accumulating neurons in the monkey cerebral cortex have a predominantly vertical organization, with GABAergic cells in deep layers providing a main source of input to cells in the more superficial layers. In the present study, a few of the axons belonging to the GABAergic cells were observed to be myelinated. Whether the other GABAergic axons are myelinated remains unknown. Smooth and sparsely spinous cells having either myelinated or unmyelinated axons, most of which ascend toward the pial surface, have been described in the visual cortex of the rat (Peters and Fairen, '78; Peters and Proskauer, '80; Peters and Rara, '851, cat, and monkey (LeVay, '73), and in layer JY of the somatosensory cortex of the mouse (Woolsey et al., '75; White, '78).

Thalamocortical synapses It has been established in a number of studies that dendrites belonging to nonspiny, nonpyramidal cells receive thalamocortical synapses (e.g., White, '78; Hornung and Garey, '81; Hersch and White, '81; White and Rock, '81; White et al., '84). White ('79, '81) has shown that a large variety of neuronal types in layer IV of the mouse barrel cortex receive direct thalamocortical synaptic input and that dendrites belonging to different types of cells receive markedly different numbers and proportions of thalamocortical synapses. This study is aimed at quantifying the input from thalamocortical and other sources to a distinct population of cortical neurons, the GABAergic cells. Prerequisite for any study of this kind is the ability to accurately identify all thalamocortical axon terminals within a given region of the cortex. In most systems, thalamocortical axon terminals degenerate over a varied time course such that at any single postlesion survival time some of the terminals are phagocytosed before others have begun to degenerate, rendering impossible the quantification of these synapses at any given survival time (eg., Tigges and Tigges, '79; Shanks and Powell, '81).In contrast, thalamocortical axon terminals in layer JY of PMBSF cortex in the mouse degenerate simultaneously and thus can all be identified after a 4-day postlesion survival time (White, '78, '79). Additional proof that all thalamocortical axon terminals in mouse PMBSF cortex are labeled by lesion-induced degen-

9

Fig. 7. GAD positive cell body (CB)forming an asymmetrical synapse with an unidentified axon terminal (A). ~35,000. Fig. 8. A GABA-positive dendrite forming asymmetrical synapses with two unidentified axon terminals (A). ~46,000.

eration is provided by a recent study in which thalamocortical axon terminals were labeled by the anterograde transport of the lectin Phaseohs vulgaris leucoagglutinin (Keller et al., '85). With this method it was established that about 20% of the asymmetrical synapses in layer IV of PMBSF cortex are formed by thalamocortical axon terminals, a figure basically identical to that obtained with lesion-induceddegeneration, This study was undertaken, in part, to establish whether GABAergic neurons form a distinct functional group with respect to their synaptic connectivity. Our results indicate that with regard to synaptic input, GABAergic neurons form a heterogeneous population: Each cell receives thalamocortical synapses, but their somata and dendrites display a marked diversity of thalamocortical and other synapses. No correlation could be established between the shape, size, or location within a barrel of these neurons and the number, type, or distribution of synapses they received. The differences in the proportions of thalamocortical axon terminals synapsing on the cells examined in this study are not related to differences in the concentration of these terminals in the surrounding neuropil but rather to some intrinsic disposition of these cells to receive specific pat-


10

Fig. 9. A lightly stained GAD-positive dendrite (asterisk) forming a n asymmetrical synapse with an unidentified axon terminal (A), and a symmetrical synapse with a GABAergic axon terminal. ~40,000. Fig. 10. A GABA-positive dendrite farming a n asymmetrical synapse with degenerating, thalamocortical axon terminal (TC).x 65,000.

terns of thalamocortical synapses. Examination of Table 1 reveals that dendrites belonging to different GABAergic neurons receive markedly different proportions of thalamocortical synapses, whereas dendrites belonging to the same neuron receive similar proportions of these synapses. The numbers and proportions of thalamocortical synapses received by dendrites belonging to the GABAergic neurons examined in this study range from 3.2 to 19.5%. It is of interest that a similar range of thalamocortical input was revealed in a study of Golgi-impregnated nonspiny multipolar cells in layer IV of the barrel cortex in the mouse (White et al., '84).

GABAergic synapses with GABA neurons An examination of Table 1reveals that each of the GABAergic cell bodies and their dendrites receives a certain proportion of their synapses from GABAergic terminals. The identity of the cells of origin of these GABAergic terminals could not be determined, but it can be assumed that these terminals originate either from other GABAergic neurons or from the same cell to which the terminals are presynaptic. Synapses between GABAergic terminals and GABAergic neurons have been documented i n several other studies (Ribak, '78; Hendry et al., '83; Hamos et al., '83). A small number of terminals, which were not labeled by either GABA or GAD, formed symmetrical synapses with GABAergic cell bodies and their dendrites. Technical problems inherent in immunocytochemistry, such as incomplete penetration of immunoreagents or antigenic "masking" make difficult the interpretation of these finding as true negatives; however, it is possible that these terminals are truly GABA or GAD negative.

Unlabeled synapses Most synapses with the GABAergic cell bodies and dendrites examined in this study are of the asymmetrical type and are made by unlabeled terminals. The sources of these terminals could not be determined but they may belong to the axon collaterals of cortical projection neurons. Blum ('74)has shown that collaterals of cortical projection neurons innervate inhibitory interneurons in the cat and monkey sensorimotor cortices, and he proposed that these connections may operate to limit repeated neuronal discharges. In a companion study, White and Keller ('87) have shown that dendrites in layer N of mouse PMBSF cortex receive synapses from terminals arising from the axon collaterals of corticothalamic projection neurons. The dendrites postsynaptic to these axon collaterals are nonspiny and in other respects resemble the dendrites belonging to the GABAergic cells examined in this study. Thus it can be assumed that a proportion of the asymmetrical synapses received by dendrites belonging to GABAergic cells are made by terminals of the local axon collaterals of projection cells.

Functional implications Results of this study indicate that every GABAergic neuron having a dendrite in layer N receives direct thalamocortical input. Based on the conclusion that every cortical neuron having a dendrite in layer IV of a primary area of the cortex forms synapses with thalamic afferents (White, '86),it can be assumed that projection cells whose dendrites pass through layer IV also receive thalamocortical synapses. In the present study it was established that in neuropil surrounding GABAergic cell bodies, approximately

GABAERGIC NEURONS 67% of the unlabeled dendrites were postsynaptic to GABAergic terminals. Since only a small number of unlabeled dendrites were examined, and these were followed for short distances, the proportion of the dendritic population in layer IV that receives GABAergic input might be even higher. From the synaptic connections described above it follows that layer IV contains a circuit in which cortical projection cells receive both thalamic and GABAergic input and that GABAergic cells receive both thalamic input and input from collaterals of projection cells. Lorente de N6 ('38) described a similar triadic circuit and suggested that it portrays the basic plan upon which the brain is organized. For a discussion of this circuit and its occurrence in other regions of the brain the reader is referred to our companion study (White and Keller, '87). Several of the synaptic patterns of GABAergic neurons observed in this study may provide a substrate for physiologically defined inhibitory processes in the cerebral cortex. For instance, Mountcastle ('57)proposed a model for twopoint discrimination of sensory stimuli in the somatosensory cortex. In this model, lateral inhibition between adjacent receptive fields serves to enhance contrast between two adjacent tactile stimuli. Pertinent to this is the observation that some neurons in the barrel cortex are inhibited when two adjacent whiskers are stimulated, thus enhancing the spatial resolution of sensory input (Simons, '83, '85). In the present study, two GABAergic cells possess dendrites that originate in one barrel and project at least as far as the hollow of a n adjacent barrel, where they are postsynaptic to thalamic afferents. These dendrites may serve as a substrate for lateral inhibition by being subject to an excitatory thalamic input in one barrel, conducting this information back to their cell bodies in a n adjacent barrel, which then, by feed-forward inhibition, suppresses other cells in that barrel that receive thalamic input. With regard to the role of GABA in lateral inhibition, Sillito ('84) suggests a cardinal role for GABA-mediated lateral inhibition in the formation of orientation selectivity and ocular dominance in the visual cortex in the cat. GABAergic cells that are postsynaptic to GABAergic terminals provide a n anatomical basis for disinhibition, which could refine and control the inhibitory actions of the GABAergic cells, via feedback inhibition. Disinhibition mechanisms have been observed in various regions of the brain, including the cerebral cortex, and their fundamental role in cerebral function has been demonstrated (Schlag and Balvin, '64;Steriade and Deschenes, '73; Kelly and Renaud, '74; Tsumoto, '78). Dykes et al. ('84) have combined extracellular recordings with pharmacological manipulations of cortical neurons in the cat primary somatosensory cortex to determine the role of GABAergic inhibition in the function of these cells. They conclude that powerful GABAergic inhibition is constantly active in the cortex, suppressing the excitation from a majority of afferent inputs that synapse on a neuron, in a way that would act to restrict receptive field sizes. In addition, thalamic stimulation induces within the cortex an afferent inhibition that dynamically manipulates receptive field properties. Dykes et al., ('84)suggest that both the tonic and afferent inhibition are mediated by the same GABAergic interneuron. Both inhibitory processes require this cell to be postsynaptic to thalamic afferents. Some of the GABAergic cells described in our study receive substantial thalamic input and so are likely candidate to function within this framework. Data from the present and previous

11 studies (e.g., Ribak, '78; Hendry et al., '83)) showing that GABAergic cells synapse preferentially onto cortical cell bodies, initial axonal segments, and dendritic branch points, indicate that these neurons are well equipped to exert powerful tonic inhibition and to shape the receptive properties of cortical neurons.

ACKNOWLEDGMENTS This work was supported by NIH grant NS 20149-04 and US-Israel BSF grant 3201/83 to E.L.W. and by a Foulkes Foundation Fellowship to A.K. We wish to thank the Roboz Surgical Instrument Co., Inc., and I.N.R., Inc., for their contributions to our research efforts. We also thank Ms. Yehudit Ganon and Ms. Smadar Levy for their valuable assistance. We are indebted to Dr. I.J. Kopin, director, Intramural Programs, NINCDS, NIH, for providing the GAD antisera.

LITERATURE CITED Blum, B.(1974) Collateral inhibition in the sensorimotor cortex of the cat and the squirrel monkey-its role in control of neuron discharges. Brain Behav. Evol. 9:237-247. Chmielowxka, J., M.G. Stewart, R.C. Bourne, and J. Hamori (1986)Gammaaminobutyric acid immunoreactivity in mouse barrel field: A light microscopical study. Brain Res. 368r371-374. Davis, T.L., and P. Sterling (1979) Microcircuitry of cat visual cortex: Classification of neurons in layer IV of area 17, and identification of the patterns of lateral geniculate input. J. Comp. Neurol. 188:599-628. DeFelipe, J., and E.G. Jones (1985) Vertical organization of gamma-aminobutyric acid-accumulating intrinsic neuronal systems in monkey cerebral cortex. J. Neurosci. 5:3246-3260. DeFelipe, J., S.H.C. Hendry, E.G. Jones, and D.Schmechel(1985)Variahility in the terminations of GABAergic chandelier cell axom on initial segments of pyramidal cell axons in the monkey sensory-motor cortex. J. Comp. Neurol. 231:364-384. Dichter, M.A. (1980)Physiological identification of GABA as the inhibitory transmitter for mammalian cortical neurons in cell culture. Brain Res. 190:lll-121. Dykes, R.W., P. Landry, R. Metherate, and T.P. Hicks (1984) Functional role of GABA in cat primary somatosensory cortex: Shaping receptive fields of cortical neurons. J. Neurophysiol. 521066-1093. Fairen, A., J. DeFelipe, and J. Regidor (1984) Nonpyramidal neurons: General account. In A. Peters and E.G. Jones (eds): Cerebral Cortex. Vol 1: Cellular Components of the Cerebral Cortex. New York Plenum Press, pp. 201-253. Feldman, M.L., and A. Peters (1974) A study of barrels and pyramidal dendritic clusters i n the cerebral cortex. Brain Res. 77r55-76. Feldman, M.L., and A. Peters (1978)The forms of non-pyramidal neurons in the visual cortex of the rat. 3. Comp. Neurol. 179761-794. Freund, T.F., K.A.C. Martin, A.D. Smith, and P. Somogyi (1983) Glutamate decarboxylase-immunoreactiveterminals of Golgi-impregnated axoaxonic cells and of presumed basket cells in synaptic contact with pyramidal neurons of the cat's visual cortex. J. Comp. Neurol. 221r263-278. Hamos, J.E., T.L. Davis, and P. Sterling (1983) Four types of neuron in layer IVab of cat cortical area 17 accumulate 3H-GABA. J. Comp. Neurol. 217:449-457. Hedlich, A., and E. Winkelmand (1982) Neuronentypen des visuellen Cortex der adulten und juvenilen Ratte. J. Hirnforsch. 23:353-373. Hendry, S.H.C.,and E.G. Junes (1981) Sizes and distribution of intrinsic neurons incorporating tritiated GABA in the monkey sensory-motor cortex. J. Neurosci. 1:390-408. Hendry, S.H.C., C.R.Houser, E.G. Jones, and J.E. Vaughn (1983) Synaptic organization of immunocytochemically identified GABA neurons in monkey sensory-motorcortex. J. Neurocytol. 12:639-660. Hersch, S.M., and E.L. White (1981)Thalamocortical synapses involving identified neurons in mouse primary somatosensory cortex: A terminal degeneration and GolgiEM study. J. Comp. Neurol. 195252-263. Hornung, J.P., and L.J. Garey (1981)The thalamic projection to cat visual cortex: ultrastructure of neurons identified by Golgi impregnation or retrograde horseradish peroxidase transport. Neuroscience 6:1053-1068.

12 Houser, C.R., S.H.C. Hendry, E.G. Jones, and J.E. Vaughn (1983) Morphological diversity of immunocytochemically identified GABA neurons in the monkey sensory-motor cortex J. Neurocytol. 12.617-638. Houser, C.R., J.E. Vaughn, S.H.C. Hendry, E.G. Jones, and A. Peters (1984) GABA neurons in the cerebral cortex. In E.G. Jones and A. Peters (eds): Cerebral Cortex. Vol 2: Cellular Components of the Cerebral Cortex. New York Plenum Press, pp. 63-69. Hsu, S.M., L. Raine, and H. Ganger (1981) The use of avidin-biotin-peroxidase complex (ABC) in immunoperoxidase techniques: A comparison between ABC and unlabeled antibody (PAP) procedures. J. Histochem. Cytochem. 29577-580. Jones, E.G. (1975) Lamination and differential distribution of thalamic afferents within the sensory-motor cortex of the squirrel monkey. J. Comp. Neurol. l60:167-204. Keller, A., E.L. White, and P.B. Cipolloni (1985) The identification of thalamocortical axon terminals in barrels of mouse SmI cortex using immunohistochemistry of anterogradely transported lectin (Phaseolus wlgaris-leucoagglutinin). Brain Res. 343:159-165. Keller, A., and E.L. White (1986) Distribution of glutamic acid decarboxylase-immunoreactivestructures in the barrel region of mouse somatosensory cortex. Neurosci. Lett. 66:245-250. Kelly, J.S., and L.P. Renaud (1974) Physiological identification of inhibitory interneurons in the feline pericruciate cortex. Neuropharmacology 13:463-474. Krnjevic, K. (1984)Neurotransmitters in cerebral cortex: A general account. I n E.G. Jones and A. Peters (eds): Cerebral cortex. Vol 2: Cellular Components of the Cerebral Cortex. New York Plenum Press, pp. 3961. Krjevic, K., and S. Schwartz (1967) The action of gamma-aminobutyric acid on cortical neurones. Exp. Brain Res. 3:320-336. LeVay, S. (1973) Synaptic patterns in the visual cortex of the cat and monkey. Electron microscopy of Golgi preparations. J. Comp. Neurol. 15Or53-86. Lin, C.S., S.M. Lu, and D.E. Schmechel(1985)Glutamic acid decarboxylase immunoreactivity in layer IV of barrel cortex of r a t and mouse. J. Neurosci. 5:1934-1939. Lorente de NO, R. (1938)Architectonics and structure of the cerebral cortex. In J.F. Fulton (ed): Physiology of the Nervous System. New York: Oxford Univ. Press, pp. 291-329. Lund, J.S. (1973) Organization of neurons in the visual cortex: Area 17, of -'the monkey (Macaca mulatta). J. Comp. Neurol. 147;455-496. Lund, J.S., G.H. Henry, C.L. MacQueen, and A.R. Harvey (1979)Anatomical organization of the primary visual cortex (area 17)of the cat. A comparison with area 17 of the Macaque monkey. J. Comp. Neurol. 184.599618. Lund, J.S., A.E. Hendrickson, M.P. Ogren, and E.A. Tobin (1981) Anatomical organization of primate visual cortex area. VII. J. Comp Neurol. 202:19-45. McMullen, N.T., and E.M. Glaser (1982)Morphology and laminar distribution of nonpyramidal neurons in the auditory cortex of the rabbit. J, Comp. Neurol. 208~85-106. Mountcastle, V.B. (1957) Modality and topographic properties of single neurons of cat's somatic sensory cortex. J. Neurophysiol. 20:408-434. Parnavelas, J.G., A.R. Lieberman, and K.E. Webster (1977)Organization of neurons in the visual cortex, area 17, of the rat. J. Anat. 124~305-322. Peters, A., and A. Fairen (1978)Smooth and sparsely-spinedstellate cells in the visual cortex of the r a t A study using a combined Golgi-electron microscope technique. J. Comp. Neurol. 181:129-172. Peters, A., and C.C. Proskauer (1980) Smooth or sparsely spined cells with myelinated axons in rat visual cortex. Neuroscience 612079-2092. Peters, A., and J. Regidor (1981) A reassessment of the forms of uonpyramidal neurons in area 17 of cat visual cortex. J. Comp. Neurol. 203:685716. Peters, A., S.L. Pafay, and H. deF. Webster (1976)The Fine Structure of the Nervous System: The Neurons and Supporting Cells. Philadelphia: W.B. Saunders. Ribak, C.E. (1978) Aspinous and sparsely-spinous stellate neurons i n the visual cortex of rats contain glutamic acid decarboxylase. J. Neurocytol. 7:461-478. Schlag, J., and R. Balvin (1964) Sequence of events following synaptic and electrical excitation of pyramidal neurons of the motor cortex. J. Neurophysiol. 27:334-365. Shanks, M.F., and T.P.S. Powell (1981) An electron microscopic study of the termination of thalamocortical fibers in areas 3b, 1 and 2 of the somatic sensory cortex of the monkey. Brain Res. 218;35-47. Sidman, R.L., J.B. Angevin, and E.T.Pierce (1971)Atlas of the Mouse Brain and Spinal Cord. Cambridge, Mass: Harvard University Press.

A. KELLER AND E.L. WHITE Sillito, A.M. (1984)Functional considerations of the operation of GABAergic inhibitory processes in the visual cortex. In E.G. Jones and A. Peters (eds): Cerebral Cortex. Vol. 2: Functional Properties of Cortical Cells. New York: Plenum Press, pp. 91-117. Simons, D.J. (1983) Multi-whisker stimulation and its effects on vibrissa units in rat SmI barrel cortex. Brain Res. 276:178-182. Simons, D.J. (1985) Temporal and spatial integration in the rat SI vibrissa cortex. J. Newophysiol. 54:615-635. Simons, D.J., and T.A. Woolsey (1984) Morphology of Golgi-Cox-impregnated barrel neurons in r a t SmI cortex. J. Comp. Neurol. 230:119-132. Somogyi, P., and H. Takagi (1982)A note on the use of picric acid-paraformaldehyde-glutaraldehyde fixative for correlated light and electron microscopic immunocytochemistry. Neuroscience 7:1779-1783. Somogyi, P., T.F. Freund, J.Y. Wu, and A.D. Smith (1983) The sectionGolgi impregnation procedure. 2. Immunocytochemical demonstration of glutamate decarboxylase in Golgi-impregnated neurons and in their afferent synaptic boutons in the visual cortex of the cat. Neuroscience 9r475490. Somogyi, P., A.J. Hodgson, A.D. Smith, M.G. Nunzi, A. Gorio, and J.Y. Wu (1984a)Different populations of gabaergic neurons in the visual cortex and hippocampus of cat contain somatostatin- or cholecystokinin-immunoreactive material. J. Neurosci. 4:2590-2603. Somogyi, P., T.F. Freund, and Z.F. Kisvarday (198413) Different types of 3HGABA accumulating neurons in the visual cortex of the rat. Characterization by combined autoradiography and Golgi impregnation. Exp. Brain Res. 54:45-56. Somogyi, P., T.F. Freund, A.J. Hodgson, J. Somogyi, D. Beroukas, and I.W. Chubb (1985)Identified axo-axonic cells are immunoreactive for GABA in the hippocampus and visual cortex of the cat. Brain Res. 332:143149. Steriade, M., and M. Deschenes (1973) Cortical interneurons during sleep and waking in freely moving primates. Brain Res. 50:192-199. Tigges, M., and J. Tigges (1979) Types of degenerating geniculocortical axon terminals and their contribution to layer IV of area 17 in the squirrel monkey (Samiri). Cell Tissue Res. 196:471-486. Tsumoto, T. (1978)Inhibitory and excitatory binocuiar convergence to visual cortical neurons of the cat. Brain Res. 159:85-97. Tsumoto, T., W. Eckart, and O.D. Creutzfeldt (1979) Modification of orientation sensitivity of cat visual cortex neurons by removal of GABAmediated inhibition. Exp. Brain Res. 34:351-363. Van der Loos, H. (1976) Barreloids in mouse somatosensory thalamus. Neurosci. Lett. 2:l-6. White, E L . (1976) Ultrastructure and synaptic connections in barrels of mouse SI cortex. Brain Res. 105:229-251. White, E.L. (1978) Identified neurons in mouse SmI cortex which are postsynaptic to thalamocortical axon terminals: A combined Golgi-electron microscopic and degeneration study. J. Comp. Neurol. 181:627-662. White, E.L. (1979)Thalamocortical synaptic relations: a review with empha' sis on the projection of specific thalamic nuclei to the primary sensory areas of the neocortex. Brain Res. Rev. 1:275-311. White, E.L. (1981)Thalamocortical synaptic relations. In F.O. Schmitt and F.G. Worden (eds): The Organization of the Cerebral Cortex. Cambridge: MIT Press, pp. 153-161. White, E.L. (1986)Terminations of thalamic afferents. In E.G. Jones and A. Peters (eds): Cerebral Cortex, Vol. 5. New York Plenum F'ress, pp. 271289. White, EL., and R. DeAmicis (1977) Afferent and efferent projections of the region in mouse SmI cortex which contains the posteromedial barrel subfield. J. Comp. Neurol. 175r455-481. White, E.L., and M.P. Rock (1981) A comparison of thalamocortical and other synaptic inputs to dendrites of two non-spiny neurons in a single barrel of mouse SmI cortex. J. Comp. Neurol. 195265-277. White, E.L., G. Benshalom, and S.M. Hersch (1984) Thalamocortical and other synapses of non-spiny multipolar cells in mouse SmI cortex. J. Comp. Neurol. 22~311-320. White, E.L., and A. Keller (1987)Intrinsic circuitry involving the local axon collaterals of corticothalamic projection neurons in mouse SmI cortex. J. Comp. Neurol. 262:13-26. Williams, M.A., and G.A.. Meek (1966) Studies on thickness variation in ultrathin sections. J. R. Microsc. SOC.85:337. Woolsey, T.A., and H. Van der Loo8 (1970) The structural organization of layer IV in the somatosensory (SI)region of mouse cerebral cortex. Brain Res. 17:205-242. Woolsey, T.A., M.K. Dierker, and D.F. Wann (1975) Mouse SmI cortex: Qualitative and quantitative classification of Golgi-impregnated barrel neurons. Proc. Natl. Acad. Sci. USA 72:2165-2169.