EXPERIMENTAL

NESJROLOGY

114,104-122

(1991)

Golgi, Histochemical, and lmmunocytochemical Analyses of the Neurons of Auditory-Related Cortices of the Rhesus monkey P. B. CIPOLLONI AND D. N. PANDYA Edith

Now-se

Rogers Memorial Veterans Anatomy and Neurology,

Administration Medical Center, Bedford, Mbssachusetts 01730; and Departments Boston University School of Medicine, Boston, Massachusetts 02118

~orpholo~cal characteristics of the neurons of the auditory cortical areas of the rhesus monkey were investigated using Golgi and horseradish peroxidase methods. Neurons of the auditory cortices can be segregated into two categories, spinous and nonspinous, which can be further subclassified according to their dendritic arrays. The spinous neurons include pyramidal, ‘&star pyramid,” multipolar, and bipolar cells. As in other cortiees, pyramidal cells are found in layers II-VI and appear to be the most numerous of all cortical neurons. The “star pyramids” have radially oriented dendrites with a less prominent apical shaft and are found mainly in the middle cortical layers. The spinous multipolar neurons are also found in the middle cortical layers and have their dendrites radially arrayed but have no apical dendrite. The spinous bipolar cells, found in the infragranular layers, occur most frequently in the lateral auditory association cortex. The nonspinous neurons include neurogliaform, multipolar, bitufted, and bipolar cells and are found in all cortical layers. The neurogliaform cells are the smallest of all neurons and have radially arrayed, recurving dendrites. The nonspinous multipolar cells also have radially arrayed dendrites but vary in size from being confined to one cortical layer to extending across four laminae. The bitufted neurons are subcl~ifi~ into three groups: neurons whose primary dendrites arise radially from their somata, those whose dendrites arise from two poles of their somata, and those that have a single primary dendrite arising from one pole and multiple dendrites from another pole of their somata. The nonspinous bipolar cells also have several variants but usually have dendrites arising from two poles of the somata. The chemical characteristics of the auditory neurons were investigated using histochemical and immunocytochemical methods. Peptidergic neurons, i.e., cholecytokinin-, vasoactive intestinal polypeptide-, somatostatin-, and substance P-reactive neurons are found in the various subregions of the auditory cortices and are distributed differentially in the cortical laminae. These neurons are of the nonpyramidal type. Gamma aminobutyric acid-reactive neurons are also nonpyramidal cells and they are found in all cortical layers. Their numbers varied among the cortical lami0014-4886/91 $3.00 Copyright 0 1991 by Academic Press, All rights of reproduction in any form

nae in the different auditory regions. The nicotinamide adenine dinucleotide phosphate-diaphor~ labeled neurons are quite numerous and are strictly of the nonpyramidal type. They are also found in all cortical laminae. Cytochrome oxidase-labeled cells are of both pyramidal and nonpyramidal types. They are distributed in the central cortical laminae and are found mainly in the auditory association areas. 0 1981 Academic pry, 1~.

I~ODU~ION Several anatomical studies have provided information on the area1 and laminar distributions of the corticocortical and thalamocortical connections of the auditory-related cortices in the monkey (7,20-22,49,50,X$ 53). The identity of the neurons receiving these inputs can be determined, however, only at the ultrast~ctural level (e.g., 6). This information could be obtained by applying methods for visualizing synapses between identified pre- and postsynaptic elements involved in these connections (26, 57, 72, 81, 82). Before applying such methods in the monkey auditory system to determine the synaptic connectivity that link auditory cortical neurons into functional groups, it is first necessary to determine the different types of neurons that occur in the auditory-related cortices. Several studies using Golgi impregnation methods have described in some detail the morphological cell types in various regions of the monkey cerebral cortex (e.g., 5,34,42,43,63,74,75). There is, however, no comprehensive analysis of the cell types of the auditory-related cortical areas. It is now possible to classify cortical neurons on the basis of their chemical specificity. Some reports have characterized cortical neurons by their neurotransmitter, such as GABAergic cells (29,30,38,64), while other studies characterized these cells by the peptides they contain, such as vasoactive intestinal polypeptide, somatostatin, substance-P, and cholecystokinin (1,5, 14, 35). With the use of histochemical stains, cortical neurons have also been segregated on the basis of intrinsic enzymes, such as cytochrome oxidase (5, 28, 86) and nicotinamide adenine dinucleotide phosphate-diapho-

104 Inc. reserved.

of

NEURONAL

ANALYSIS

rase (18, 51, 65). Although several investigators have characterized populations of chemically labeled neurons in various areas of the monkey cerebral cortex (see (14) for review), few studies have described their distributions in the auditory cortices (4). The aim of the present study is to describe, with the aid of several Golgi techniques and the horseradish peroxidase method, the various neuronal types of the auditory-related co&ices of the monkey. In addition, we will present preliminary observations regarding some of the chemical characteristics of the neurons in these cortical areas using histochemical and immunocytochemical methods. METHODS

Twenty adult rhesus monkey brains were used for this study. Thirteen animals used for the silver impregnation studies were deeply anesthetized with sodium pentobarbital prior to perfusion. These animals were perfused transcardially with normal saline followed by 10% buffered formalin solution. The brains were then stored in 10% buffered formalin after each hemispheric surface was photographed. The superior temporal gyrus (STG), including the supratemporal plane, was removed en bloc at the depths of the lateral fissure and the superior temporal sulcus (Fig. 1A). In each case the superior temporal region was divided transversely into six blocks. Some of the blocks were cut into 120 pm sections prior to silver impregnation with the slice-Golgi method (33). Other blocks were silver impregnated according to the procedure of Braitenberg et al. (3) or the rapid-Golgi method of Valverde (74). Subsequently, these blocks were cut coronally into lOO- to 120-&m sections with a vibratome. All impregnated tissue sections were mounted serially onto glass slides with Entellan. Three animals received a series of injections of horseradish peroxidase (HRP) in area Ts3 of STG (Fig. 1) to label neurons in the adjoining area paAh (22). Each injection contained 0.05 ~1 of a 30% solution of HRP. After 1 to 3 days survival, these animals were transcardially perfused with 0.5% paraformaldehyde and 2.0% giutaraldehyde in a buffered 0.1 M phosphate solution. After photographing the hemispheres, selected blocks i Abbreviations: AS, arcuate sulcus; CS, central sulcus; IPS, intraparietal sulcus; KA, koniocortex; Kalt, lateral koniocortex; Kam, medial koniocortex; LS, lunate sulcus; paAc, caudal parakoni~o~ex; paAlt, lateral parakoniocortex; paAr, rostra1 parakoniocortex; pa1, parainsular cortex; pAI1, periallocortex; Pro, proisocortex; proA, auditory prokoniocortex; pros, somatosensory prokonioeortex; PS, principle sulcus; reIpt, retroinsular parietotemporal cortex; reIt, retroinsular temporal cortex; SF(LF), Sylvian fissure (lateral fissure); STG, superior temporal gyrus; STR, superior temporal region; STS, superior temporal sulcus; Tpt, temporoparietal cortex; Tsl, Ts2, Ts3, temporalis superior cortex 1, 2, 3; TPO, multimodal cortex of the superior temporal sulcus.

OF

AUDITORY

CORTEX

105

of the auditory cortices were cut into lOO-pm-thick sections with a vibratome and reacted according to the protocol of Itoh et al. (32) for HRP histochemistry. Other sections from these blocks were Golgi-impregnated according to the single-slice method of Izzo et al. (33). The material from both the Golgi impregnations and the HRP histochemistry was examined with the light microscope. Selected neurons were drawn with the aid of a drawing tube. The widest diameters of the somata of the various neuronal types were measured in the material prepared with the rapid Golgi technique of ValVerde (74). Four animals were used for histochemical and immunocytochemical analyses. One of these animals was perfused under deep nembutal anesthesia with 0.5% paraformaldehyde and 2.0% glutaraldehyde and used only for histochemical studies. The brains of three animals were removed fresh after nembutal overdose and immersion-fixed in a solution of 2% PLP (periodate, lysine, and paraformaldehyde) in a neutral phosphate buffer (45). Selected blocks from the perfusion-fixed brain were cut with a vibratome or freezing microtome and reacted for the nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) enzyme (18) or for cytochrome oxidase (CO) activity (12, 85, 86). Specifically, in order to stain the NADPH-d-reactive neurons, sections were incubated in a solution of 100 mg% nitroblue tetrazolium, 50 mg% NADPH (type l), 125 mg% monosodium malate, and 0.8% Triton X-100 in 0.1 M phosphate buffer, pH 8.0, at 37°C. After staining, the sections were washed in 0.1 M phosphate buffer at pH 7.3 and mounted on chrome-gel-coated slides. Sections treated by heating or by reacting without NADPH in the solutions served as controls. For cytochrome oxidase staining, free-floating sections were incubated in a solution consisting of 30 mg% cytochrome c, 50 mg% 3,3’diaminobenzidine tetrahydrochloride (type 2), and 20 mg% catalase in 0.1 Mphosphate buffer (pH 7.3) in the dark at 37°C for 6 h. The sections were then washed in 0.1 M phosphate buffer and mounted on chrome-alumcoated slides. Pretreatment of some sections with potassium cyanide served as controls. The immersion-fixed brains were cut with a freezing microtome and immunocytochemically labeled with the aid of polyclonal antibodies (61) for gamma aminobutyric acid (Immunonuclear, Stillwater, MN), somatostatin (courtesy of L, Chun, Dept. of Neurology, Massachusetts General Hospital, Boston, MA), substance-P ~Immunonu~lear), cholecystokinin (Immunonu~lear), and vasoactive intestinal polypeptide (Immunonuclear). Immunocytochemistry was performed on 50-pmthick sections of the auditory-related cortices. Tissue sections were preincubated in a solution of absolute methanol and 0.3% hydrogen peroxide for 30 min, washed in three changes of phosphate-buffered saline (PBS) (pH 7.4) for 10 min each, placed in 10% normal

106

CIPOLLONI

AND

PANDYA

C FIG. 1. (A) Diagram to show the boundary of the superior temporal region on the lateral surface of the cerebral hemisphere. The Sylvian fissure is opened to expose the insula, the circular sulcus (CiS), and the superior temporal plane (STP). (B) Diagram of opened Sylvian fissure and superior temporal sulcus to show topographic locations of the architectonic areas of the superior temporal region (STR) according to Galaburda and Pandya (83). (C!) Diagram showing the architectonic areas of STR arranged according to three lines: the root line of the circular sulcus (CXS), the core line of the superior temporal plane (SW), and the belt line of the superior temporal gyrus (STG). This diagram also shows four stages in the rostrocaudal direction for the architectonic areas of STR. Each stage contains an area from the root, core and belt lines.

goat serum (GIBCO Labs, Grand Island, NY) for 1 h, incubated free-floating in the selected primary antiserum to GABA or each of the neuropeptides separately at room temperature for 12-18 h (1:lOOO dilution with 0.3% Triton X-100 and 10% normal goat serum), washed again in three changes of PBS for 10 min each, placed in peroxidase-conjugated goat antirabbit IgG (1:300 in PBS) (Boerhinger-Mannheim, Indianapolis, IN), washed in three changes of PBS for 10 min each, and reacted with 3,3’-diaminobenzidine HCL (1 mg/ml) in Tris-HCL buffer with 0.005% hydrogen peroxide in the dark at 37°C for 6 h. In all cases, silver intensification of the reaction product was done according to the method of Gallyas et al. (23). The sections were again washed with three changes of PBS for 10 min each and mounted on chrome-alum-coated slides. As controls for the specificity of immunostaining for CCK and GABA, tissue sections were incubated in PBS or in preimmune serum without primary antiserum and were found to have no peroxidase reaction product. As controls for SS and SP immunostaining, the antibodies

to these peptides were preadsorbed with an excess of the specific peptides prior to incubation of the tissue sections. The laminar and area1 distributions of the labeled neurons were determined from adjacent sections prepared with Nissl stain. Distribution patterns of the histo- and immunocytochemically labeled neurons in representative regions of the auditory-related cortices were mapped with the aid of a drawing tube. Selected labeled neurons were then photographed. RESULTS

The architectonic parcellation and the nomenclature for the subregions of the auditory-related cortices used in this report (Fig. 1) are according to those of Galaburda and Pandya (22). We have limited our observations to the primary auditory region (area KA) and to the surrounding auditory-related areas Pro, proA, and pa1 in the circular sulcus (CiS); areas paAr and paAc in the supratemporal plane (STP); and areas Ts2, Ts3,

NEURONAL

ANALYSIS

OF

AUDITORY

CORTEX

107

paAlt, and Tpt in the superior temporal gyrus (STG). For the morphological classification of the nonpyramida1 neurons we have followed the nomenclature of Feldman and Peters (17). Go&i and HRP Analyses The classification of neurons is usually based on the morphology and spatial ~stribution of t.heir dendrites and somata as well as the occurrence of spines. In some studies, the morphology and ramification patterns of axons have provided an additional means for classifying neurons (56). In our material, however, the impregnation of axons was usually not adequate to differentiate various neuronal subgroups on the basis of axonal arrays (c.f., 43). Except for minor variations, the distribution of Golgi-impregnated neurons was similar in all the auditory cortices. The dendritic morphology for HRP-labeled cells was basically the same as that for similar neurons identified with Golgi impregnation. Likewise, the morphologies of NA~PH-d-stained nonpyramidal cells were similar to those that were Golgi impregnated. The laminar locations of representative labeled and impregnated neurons are illustrated in the composite Figs. 2 and 3. Golgi-impregnated neurons were observed in all cortical layers of the auditory-related cortices. The laminar designation of the neurons refers to the location of their somata although the dendrites of many of the neurons extended into other layers, i.e., the dendrites of many of the neurons of layers II-VI were traced into layer I. Only a few of the layer I neurons per se, however, were impregnated in our preparations. The neurons in this study are segregated into two major groups, namely, spinous and nonspinous. Nonspinous neurons, however, may have a few spines on their dendrites and/or somata. The auditory cortical neurons may also be classified as pyramidal and nonpyramidal, based on the morphology of their somata and the distribution of their dendrites. Most nonpyramidal types have few to no spines on their dendrites and/or somata and are thus termed nonspinous. Other nonpyramidal types and all the pyramidal cells are termed spinous. Since Golgi impregnation of neurons is inconsistent, it was difficult to reliably discern quantitative differences in area1 and laminar distributions of impregnated neurons among the subregions of the auditory-related cortices. Only general distribution patterns for these neurons, therefore, can be described. Spinous neurons. Throughout the auditory cortices, the most numerous of the spinous neurons are pyramidal cells. These neurons are found in all cortical layers except layer I (Fig. 2). Layer II, upper layer III, and layer IV contain small (16-32 pm)- to medium (33-40 pm)sized pyramids while the largest of this cell type (41-72 pm) are found in lower layer III, layer V, and layer VI.

A collage of representative spinous neurons from the FIG. 2. auditory-related cortices labeled by retrograde filling with horseradish peroxidase (Cells A, C, E, H, and I), NADPH-diaphorase stain (Cell Y) and Golgi impregnation (all other cells) drawn with the aid of a camera lucida. Cells K, M, N, and P are examples of “star” pyramids; cells 0, R, S and Y are examples of spinous multipolar neurons; cell W is an inverted pyramid; and cells U and X are spinous bipolar neurons. All other cells are examples of the various mo~holo~es of the pyramidal neurons typically seen in the auditory cortex. The cortical layers are indicated on the left. (Arrows indicate axons. Bar indicates magnification.)

Their round- to pyramidal-shaped somata usually give rise to a single apical dendrite that has tangential branches as it courses towards the pia, In some cases, two prominent apical shafts arise from the soma (Fig. 2D) or a single apical dendrite arises from the soma and bifurcates soon after its origin (Figs. 2E and 2G). All pyramidal cells have several dendrites forming basal skirts. The axon belonging to these neurons originates from the base of the cell body or from the proximal segment of a basal dendrite and gives rise to local collaterals as it courses toward the white matter (Figs. 2A, 2B, 2Q, and 2V). In those instances in which layer II and III pyramidal neurons in the lateral auditory association cortex (area paAlt) were retrogradely labeled by horseradish peroxidase injections, more complete visualization of the axons was achieved (Figs. 2A, ZE, 2H, and 21). Some of the smallest spinous neurons have dendrites radially ~st~buted around the cell body (8-10 pm). The

108

CIPOLLONI

I II -

Ill

IV w VI FIG. 3. A collage of representative nonspinous neurons labeled by Golgi impregnation drawn with the aid of a camera lucida. Cells A, J, M, N, and Q are examples of multipolar neurons; cells C, F, I, 0 and V are examples of bitufted neurons; and cells B, D, E, G, H, K, P, R, T, and U are examples of bipolar neurons typically seen in the auditory cortex. Cells L and S are examples of a transition form of the latter two types termed unit&ted-unipolar neurons. The cortical layers are indicated on the left. (Arrows indicate axons. Bar indicates magnification.)

dendritic trees of these neurons are confined to one or two cortical layers, usually layer IV, and these neurons have less prominent apical dendrites than do typical pyramidal cells (Figs. 2K, 2M, 2N, and 2P). These neurons are similar to the “star pyramid” previously described by Lorente de No (42) and are very numerous in the middle cortical laminae. Except for these “star pyramids,” the apical dendritic shafts of most impregnated pyramidal cells extend up to layer II with the majority extending into layer I. Inverted pyramidal neurons are occasionally seen (Fig. 2W). They have all the morphological features of typical pyramidal neurons but are oriented such that their “apical” dendrites course toward the white matter whereas their axons arise from the pial aspect of the cell body. An additional spinous neuronal type, the spinous bipolar neuron (Figs. 2U and 2X), is observed mainly at the lateral border of the auditory association cortex (area paAlt). Their somata (lo-20 pm) are found in layers IV-VI. This cell type is characterized by a prominent superficial dendrite coursing toward the pia per-

AND

PANDYA

pendicular to the cortical layers and by an equally prominent basal dendrite coursing obliquely toward the white matter. Their axons arise from the base of the soma and course toward the white matter, like those of typical pyramidal neurons. Another type of spinous neuron seen infrequently in the monkey auditory cortex is the spinous multipolar cell (Figs. 20, 2R, 2s and 2Y). It is characterized by a round cell body (33-40 pm) and radially arrayed spinous dendrites. The axon, which ramifies locally, may arise from any portion of the soma or proximal portions of the dendrites. Although spinous multipolar cells can be found in other cortical layers, they are usually observed in layer IV and somewhat less frequently in layer V. Nonspinous neurons. The nonspinous neurons in these cortices occur in all cortical layers and, in general, have round to oval somata and beaded dendrites. Several morphological types of nonspinous neurons were identified with the Golgi and NADPH-diaphorase methods. The smallest of these are the neurogliaform cells (4-8 pm) which have numerous smooth, short dendrites that may recurve on themselves (Fig. 12A). The axons of these cells seem to be tightly entwined among the dendrites. This neuronal type is usually observed in layers II through V. A second type of nonspinous neuron is a multipolar cell (Figs. 3A, 35,3M, 3N, 3Q, 12B12D), which is larger than the neurogliaform cell. These nonspinous multipolar neurons have round somata (30-50 pm) and radially arrayed dendrites. The sizes of their dendritic fields vary considerably so that the fields may be limited to one cortical layer or may extend across as many as four layers. Cell bodies of these neurons occur in all cortical layers. Another type of nonspinous cell is the bitufted neuron, which has two tufts of dendrites typically arising from two poles of a round to oval shaped cell body (lo-40 pm) and are found in all cortical layers (Figs. 3 and 12). Their dendrites are usually vertically arrayed but they may be horizontally oriented, especially in layers V and VI (Fig. 3V). The bitufted cells can be classified into three subtypes: one subtype has dendrites that are arrayed in tufts even though they arise radially from the somata (Figs. 3C, 31, and 12E), a second subtype has similar dendritic arrays, but the multiple primary dendrites arise from the two poles of their somata (Figs. 3F, 3V, and 12F), and a third subtype in which the dendrites of one tuft arise from a single primary dendrite which branches into the secondary dendrites to form a “unit&ted-unipolar” dendritic array (Figs. 3L, 3S, and 12G). Bipolar cells constitute another type of nonspinous neuron (Figs. 3G, 3K, 3P, 3R, and 121). These neurons are relatively small and have round- to spindle-shaped cell bodies (12-19 pm) with single primary dendrites from both poles each giving rise to several secondary dendrites arrayed in a narrow hourglass distribution. They are usually verti-

NEURONAL

ANALYSIS

OF

AUDITORY

CORTEX

109

tally oriented in the cortex, but they may be oriented parallel to the pial surface (Figs. 3B, 3E, 3H, and 3U). The bipolar type also has several variants (Figs. 3D, 3E, 3G, 3H, 3K, 3P, 3R, 3T, 12H, and 121). All the nonspinous neuronal classes have one or more axons which may arise from either the soma and/or the proximal dendrites. None of the axons of the nonpyramidal neurons extended into the white matter. Although the nonspinous neurons can be routinely segregated into four major types on the basis of their dendritic and somatic morphologies, transition forms between these types, as described above, are also common. Indeed, the morphologies of the nonspinous cells can be viewed as a spectrum (see Fig. 12). Cytochemical

Analysis

To characterize further the neurons of the auditoryrelated cortices, we used two cytochemical techniques to demonstrate some of the neurochemicals and a neurotransmitter contained in subclasses of these neurons. Neuronal subpopulations were labeled immunocytochemically for gamma-aminobutyric acid (GABA) and for several peptides, such as substance P (SP), vasoactive intestinal polypeptide (VIP), somatostatin (SS), and cholecystokinin (CCK). In addition, neurons reactive for cytochrome oxidase (CO) and nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) were labeled with histochemical methods. Except for the complete labeling obtained with the NADPH-d method, the chemical methods labeled only somata and/or proximal dendrites. With this degree of labeling, segregation of neurons into spinous and nonspinous types was possible, however, further segregation into subtypes was not consistently possible for the nonspinous cells. In contrast to the Golgi preparations where there is no discernible differential ~stribution of the neuronal types among the architectonic areas of the supratemporal region (STR), the chemically labeled neurons are differentially distributed in these subregions. In describing the distributions of these chemically labeled neurons, we continued to follow the organization of the architectonic sub~visions of the au~to~-related cortical areas as described by Galaburda and Pandya (22), according to whom the STR is subdivided into three main architectonic lines: the root, core, and belt lines (Fig. 1). The root line, located in the circular sulcus, culminates in the second auditory area AI1 (area proA). The core line, located in the STP, leads to the koniocortical area KA, the primary auditory region, The belt line, located in the STG, is considered the cortical auditory association region. GABAergic neurons were found in all layers of all cortical divisions. These neurons appeared to be nonpyramidal only and had round to oval somata (Fig. 4).

FIG. 4. Light micrographs of representative areas showing the distribution of GABA-labeled neurons in areas KA (A) and paA& (B). The cortical layers are indicated on the lefi of each micrograph and selected GABAergic cells are indicated with arrowheads (Magnification Bar = 0.1 mm.).

Layers I, V, and VI had few GABAergic neurons while layers II and IV contained dense populations of these cells. The density of these cells in layer III was intermediate. There was no apparent difference between the primary auditory cortical regions (core line) as compared to the association auditory cortical areas (belt line) in terms of the distribution of these neurons except that the overall density of the GABAergic neurons was greater in area KA than in area paAlt (Fig. 4). No GABAergic neurons were seen in the white matter underlying these auditory cortical areas. As shown in Fig. 5A, SP-reactive neurons were observed in various subdivisions of the STR. Although only the somata and very proximal dendrites of the SPreactive neurons were labeled, they appeared to be mainly of the bipolar, bitufted, and multipolar nonspinous types (Figs. 5B and 5C). The distribution of the SP-positive neurons was different in the rostra1 auditory-related cortical areas as compared to the caudal areas and in the subregions of the three architectonic

CIPOLLONI

AND

PANDYA

FIG. 5. Schematic drawings of partial transverse sections of STR to illustrate the laminar and area1 distribution patterns of substance-P (SP)-reactive neurons in the auditory cortices. In this figure and Figs. 5-8, the section on the left is rostra1 in location and that on the right is from a caudal location in STR (Refer to Fig. 1). The micrographs show SP-reactive neurons and fibers. In this and the subsequent six figures the arrowheads indicate examples of the labeled neurons and the magnifications bars are all equal to 0.1 mm.

lines of the STR. Specifically, the rostra1 root line area Pro had relatively few SP-positive neurons and these were found in layers III, V, and VI. The core line region had substantial numbers of these neurons in area Ts2 and area paAr. In these two areas, SP-reactive neurons were distributed preferentially in layers II and III with

fewer in layer IV. Only a few labeled neurons were found in layers V and VI. The distribution of SP-positive cells in the belt line area Ts3 was mainly in the layers II and III. In the caudal auditory-related cortices, however, the root line areas proA and pa1 had relatively fewer SPpositive cells than the core line area KA and were found

NEURONAL

ANALYSIS

predominantly in the lower part of layer III and in layer V. In the core line, substantial numbers of these neurons were found in layers II and III and only occasionally in layers V and VI. These neurons in the core line were mainly in the medial portion of the primary auditory region. The lateral part of the core line was devoid of SP-positive cells. The caudal belt line regions (areas paAlt and Ts3) also had a number of these cells. They were distributed mainly in layers II and III with few in infragranular layers. At the border of the various architectonic regions there was a marked paucity of SP-reactive cells. The VIP-reactive neurons in the auditory-related cortices appeared to be nonpyramidal and were few in number (Figs. 6B and 6C). They were found mainly in the root line and the most medial part of the primary auditory region (area KA of the core line). Only occasional VIP-reactive cells were seen in the belt line. In all areas, they were located primarily in layers II and III (Fig. 6A). The number of SS-reactive neurons in the auditoryrelated areas was considerably less than the number of SP-reactive neurons (Fig. 7A). In the root line regions (areas Pro, pa1, and proA), these cells were found in layers II and III and occasionally in layers V and VI. In the more rostra1 areas, they were more numerous in the infragranular layers. The rostra1 core line regions (areas Ts2 and paAr) had SS-reactive cells which were mainly in layers II and III with some in layers V and VI. The caudal core line area KA had only occasional SS-reactive cells which were in layer II. In the belt line association regions (areas Ts2, Ts3, and paAlt) these neurons were in layers II and III. The SS-reactive cells were more completely labeled as compared to the SP-reactive ceils and were of the nonpyramidal types including nonspinous multipolar, bitufted, and bipolar neurons (Figs. 7B-7D). The CCK-reactive neurons in the auditory-related cortical areas were sparsely distributed in root and core as well as belt line regions and were mainly in the supragranular layers (Fig. BA). Like the other peptidergic neurons, they also appeared to be of the nonpyramidal type (Fig. 8B). Cytochrome oxidase-positive cells also occurred in root, core, and belt line regions (Fig. 9A). In the root line (area Pro) these neurons were distributed sparsely and only in infragranular layers V and VI. In the core line (areas paAr and KA), the labeled neurons were predominantly in the infragranular layer Va but some were also in the lower portion of layer III. In the belt line (area paAlt) they were mainly in supragranular layer 111~ and infragranular layer V. These. neurons were usually medium to large pyramidal cells but, occasionally, nonpyramidal types were also seen (Figs. 9B and 9C). The NADPH-d positive cells (Fig. 10) were found cliffusely throughout the auditory-related cortices in the

OF AUDITORY

111

CORTEX

circular sulcus (root line), the supratemporal plane (core line), and the superior temporal gyrus (belt line). The NADPH-d method densely stained the cell bodies, dendrites, and axons (Fig. 11). These cells were clearly nonpyramidal in type and, while nonspinous multipolar, bitufted and bipolar neurons were seen, most of them were multipolar and were found throughout all layers (Fig. 11). Vertical bipolar and bit&ted neurons were also labeled, but they were less numerous (Fig. 1lC). Norizontal nonspinous bipolar cells were seen in layers V and VI (Fig. 11B). The subcortical white matter also had numerous NADPH-d reactive cells that were mainly multipolar. The frequency and laminar distribution of these cells had differential patterns within the circular sulcus, STP, and STG. The root line areas had these cells in both the supra- and infragranular layers but predominantly in the latter. The belt line regions had NADPH-d reactive cells also predominantly in infragranular layers V and VI with some in supragranular layer III. The core line regions, however, had reactive cells in both the supra- and infragranular layers in almost equal proportions. Those in the supragranular layers were mainly in layer III and those in the infragranular layers were in both layers V and VI. DISCUSSION

Several studies using Golgi techniques have described the various neuronal cell types in the cortices of primate and nonprimate species (e.g., 34,43,46,47,56,59,63,67, 84). These cell types have been characterized on the basis of the mo~holo~ of their somata, axonal arbors, and dendritic configurations as well as the relative density of their spines. The main purpose of the present report is to characterize and localize the various types of neurons in the auditory-related cortices of the rhesus monkey and to correlate these observations with findings obtained with histo- and immunocytochemical labeling techniques. In our study, although the rapidGolgi, Braitenberg, and slice-Golgi methods have allowed the clear delineation of most of the neuronal characteristics, the axonal processes were not sufficiently imprecated to be used for the classification of cell types (43). The neurons of the auditory cortices are subdivided into two broad categories, i.e., spinous and nonspinous. Neurons in each of these two categories are further subdivided on the basis of the configuration of their dendritic trees. According to our observations, the most numerous of all impregnated spinous neurons are the pyramidal type (Fig. 2). In the auditory cortices of the monkey, the pyramidal neurons have the same morphological characteristics as those in other cortices of primate and nonprimate species (16). In order to understand the ~nctional role of the pyramidal neurons one must consider their efferent and afferent connectivity.

~IPOLLONI

AND I’ANDYA

i. \ ‘J

lnsula

FIG. 6. Schematic drawings of partial transverse sections of STR to illustrate the laminar and area1 distribution patterns of vasoactive intestinal polypeptide (VIP)-reactive neurons in the auditory cortices. The micrographs show VIP-reactive neurons and fibers.

For instance, cortical layer III pyramids are known to have projections to ipsi- and contralateral association areas with a few projecting to subcortical structures. Layer V and VI pyramidal neurons, in contrast, project predominantly to subcortical structures and have limited cortical projections. Pyramidal cells also have extensive local axonal projections (16). The afferent connections of these cells can be demonstrated with ultrastructural methods. For example, pyramidal cells have

been shown to receive thalamic input in rodents (57,77, 79), cat (27,31), and monkey (8). Furthermore, cailosal afferents have been demonstrated to contact pyramidal neurons in the rat (9), cat (31), and monkey (Cipolloni and Pandya, unpublished results). Pyramidal neurons also receive local association afferents (39) as well as intrinsic connections (13,71). Thus, the pyramidal neurons of the neocortex play a role in both extrinsic and intrinsic cortical connectivity.

NEURONAL

FIG. 7. (SS)-reactive

Schematic neurons

drawings of partial transverse in the auditory cortices. The

ANALYSIS

OF

AUDITORY

sections of STR to illustrate micrographs show S&reactive

Unlike the typical pyramidal neuron, the “star pyramid” (Figs. 2K, 2M, 2N, and 2P) is smaller and is characterized by more radially arrayed basal dendrites and a less prominent apical dendrite so that the dendritic field tends to be confined to one or two cortical layers (42). These are most commonly found in the middle cortical laminae in the monkey and in other species and may be a transition form between the typical pyramidal neuron and the spinous stellate cell (34). From our observations, a significant number of the “granule” cells of layer IV are “star pyramids”. These have been de-

113

CORTEX

the laminar and area1 distribution neurons and fibers.

patterns

of somatostatin

scribed as projecting only locally and are thus classified as interneurons (44). The spinous bipolar cells (Figs. 2U and 2X), typically found in layers IV-VI of the lateral auditory association cortex (area paAlt), appear similar to an atypical pyramidal cell type described by Globus and Scheibel (24) in the visual cortex of the rabbit. Spinous multipolar neurons are infrequently observed in the auditory-related cortices (Figs. 20,2R, 2S, and 2Y). Typically this neuronal type is observed in layer IV and less commonly in layer V. The spinous multipolar neurons in these cortical regions of the mon-

114

CIPOLLONI

FIG. 8. kinin

Schematic (CCK)-reactive

AND

drawings of partial transverse sections of STR neurons in the auditory cortices. The micrograph

key generally resemble the spinous multipolar neurons described in other neocortical areas of the primate and in other species (44,83). Other investigators have shown that the axons of this cell type project only locally ex-

PANDYA

to illustrate the laminar shows (CCK)-reactive

and area1 distribution neurons and fibers.

patterns

of cholecysto-

cept for some in the visual cortex of the monkey (44). Several ultramicroscopic studies have shown these cells to receive thalamocortical afferents in other species (57, 77,79,83). It is not known, however, whether these af-

NEURONAL

FIG. oxidase

9. Schematic (CO)-labeled

ANALYSIS

drawings of partial transverse sections of STR neurons in the auditory co&ices. The micrographs

ferents synapse with the spinous multipolar neurons in the auditory cortex of the primate. The nonspinous neurons in the cerebral cortices of the monkey (e.g., 34,43) and other animals (e.g., 1547, 55, 59, 60, 67, 84) have been described extensively. According to our observations, the nonspinous neurons in the auditory cortices of the monkey can be divided into four types: neurogliaform, multipolar, bit&ted, and bipolar cells (Figs. 3 and 12). The neurogliaform cells are seen in cortical layers II through VI. Ran&n y Cajal

OF

AUDITORY

CORTEX

to illustrate the laminar and area1 ~st~bution show CO-labeled neurons and fibers.

patterns

of cytochrome

described this neuron as “arachniforme” in the cat (11, 63) and human visual cortices (62). Peters and Regidor (59) classified this cell type as a small multipolar neuron in the cat visual cortex, while other investigators have termed this neuronal type as cells with glomerular axons (41), web-like cells (21, class II-small stellates (24), and “clewed” cells (76). In the monkey, this neuronal type has been described in the visual (43, 73, 751, somatosenso~ (341, and entorhinal (5) co&ices. Although specific information on the connectivity of neur-

116

CIPOLLONI

AND

PANDYA

FIG. 10. Schematic drawings of partial transverse sections of STR to illustrate the laminar and area1 distribution patterns of nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-diaphorase)-labeled neurons in the auditory cortices and the subcortical white matter. The section on the top left is the most rostra1 and that on the bottom right is most caudal (Refer to Fig. 1).

ogliaform cells is not available, it has been postulated that in the cat visual cortex they may be a major recipient of thalamic input (59). Apparently, the most common nonspinous cell is the multipolar neuron, which has a round cell body giving rise to radially arrayed dendrites similar to the spinous multipolar neuron. Unlike the spinous multipolar neurons, however, these neurons are commonly found in cortical layers II through VI and may vary considerably in size. Another type of nonspinous neuron seen in the auditory-related cortices of the monkey is the bitufted cell. These neurons are observed in all cortical layers. Based on their dendritic arrays, we have subclassified them

into three groups (see above). Their axons are found to arise from any portion of the somata or proximal dendrites. Because of distinctive axonal arrays, several investigators have identified some of these neurons, as well as some of the nonspinous multipolar cells, as “chandelier” cells (54,58). Subgroups of the nonspinous bitufted and nonspinous bipolar neurons have been described as “double bouquet” cells based again on the distribution of their axonal plexuses (63, 70). Differences in the morphologies of various types of nonspinous neurons, especially of the bit&ted and bipolar types, are not always definitive. Thus, it seems reasonable to consider all the nonspinous types as a morphological continuum. For example, as shown in Fig. 12,

NEURONAL

ANALYSIS

OF

AUDITORY

FIG. 11. These micrographs are representative of the NADPH-diaphorase neurons ’ nonspinous multipolar and bitufted neurons (magnification X165). (B) An example ncJnspinous bitufted neuron. 01

CORTEX

117

and fibers seen in the auditory cortices. (A) Examp lies of a nonspinous bipolar neuron. (C) An example ctf a

118

CIPOLLONI

A

B

C

AND

PANDYA

E

F

G

H

I

FIG. 12. Drawings of Golgi-impregnated nonspinous neurons arranged so as to illustrate the morphological continuum of the dendritic distributions of these neurons seen in the auditory cortices. (A) Neurogliaform. (B) Small multipolar. (C) Medium-sized multipolar. (D) Large multipolar. (E) Bitufted with dendrites arising radially. (F) Bitufted with dendrites arising from two poles. (G) Unitufted-unipolar. (H) Bipolar with one widespread dendritic array. (I) Bipolar with narrow dendritic arrays. (Bar indicates magnification.)

at one end of the spectrum are the neurogliaform cells (Fig. 12A) which are followed by the multipolar neurons (Figs. 12B-12D). Both of these cell types have spherical somata which have radially originating dendrites that are distributed in radial patterns. Progressively along the continuum are the type of bitufted cells which have spherical somata with dendrites distributed in two bundles even though they arise radially from the cell bodies (Fig. 12E). A further stage along this line would be the classical bit&ted neurons, which have multiple dendrites originating from either pole of the somata and are also distributed in two bundles (Fig. 12F). The next step would be a transition form seen occasionally, the “unipolar-unitufted” type, which characteristically has multiple dendrites originating from one pole of the soma and a single dendrite arising from the other pole (Fig. 12G). These dendrites then give rise to multiple branches. Further along in this spectrum is another neuronal type, the bipolar cell (Fig. 121), which has single dendrites arising from either pole of the soma. A transition form of this cell type also has two primary dendrites (Fig. 12H). One of its primary dendrites subdivides into multiple secondary branches that are arrayed in a tight bundle, whereas, the dendritic array of the other is more widespread. Finally, at the end of this spectrum is the more common bipolar cell whose dendritic arrays are distributed in narrow bundles (Fig. 121). Although it is tempting to arrange these neurons in such a linear continuum starting with the neurogliaform cell and proceeding to the bipolar cell, it must be emphasized that this arrangement is made strictly on the basis of somal and dendritic characteristics. It could be that multipolar, bitufted, and bipolar cells have no such hierarchical relationships and each is developmentally independent.

Given the current state of knowledge of cortical circuitry at the ultrastructural level, it is now possible to make certain assumptions about the roles of the various neurons in this circuitry based on their morphological features. For instance, all corticocortical and thalamocortical afferents form asymmetrical synapses with their postsynaptic cells. The portions of the postsynaptic neurons that receive this extrinsic input can be predicted on the basis of the morphology of these postsynaptic cells since it is known that spinous neurons form asymmetrical synapses on their dendrites and spines but not somata, and nonspinous neurons form asymmetrical synapses on both their dendrites and somata (80). Extrinsic afferents may therefore form synapses with dendrites and spines of all neuronal types and with the somata of nonspinous neurons but not with the somata of spinous neurons. The distribution of the extrinsic afferent contacts on the neuronal elements, otherwise, appears to be nonspecific in that any postsynaptic profile in the terminal field that is capable of forming asymmetrical synapses can receive these afferents (79). Thalamocortical afferents, for instance, have been shown to contact, in addition to spines and dendrites of spinous neurons, the somata and dendrites of multipolar, bitufted, and bipolar neurons in various systems in rodents (57,80), cats (31), and monkeys (6) without apparent regard to the configuration of their dendritic trees. Secondly, it is also known that nonpyramidal neurons usually have only local axonal projections while pyramidal cells project both locally and extrinsically. The majority of the axons of the nonspinous neurons also form symmetrical synapses, which are thought to be inhibitory, while the axons of the spinous neurons usually form asymmetrical synapses, which are thought

NEURONAL

ANALYSIS

to be excitatory (80). The morphology of the axonal arborizations of some of the nonpyramidal neurons can also give clear indication of the neuronal profiles postsynaptic to their axons such as is the case with “chandelier” cells which project only to the initial axonal segment of pyramidal neurons to form symmetrical synapses (54, 58). Thus, certain aspects of a neuron’s functional role in cortical circuity can be predicted on the basis of its morphology. In addition, knowledge of the chemical constituents of identified neurons can provide insight into their function. A number of investigations in recent years have demonstrated that subpopulations of neurons can be segregated on the basis of their histo- and immunocytochemical reactivity (14, 18,40). The functional role of some of these chemicals, such as GABA (69) and CO (86) is known but the exact functional roles of others, such as the neuropeptides and NADPH-d, remain to be determined. We have identified the various neuronal types that contain some of these substances in the auditory cortex of the monkey. GABAergic neurons, for instance, were found in all cortical layers and must have an inhibitory role in intrinsic neuronal circuitry (30,69). The distribution of these neurons differed in the various cortical layers (Fig. 4). From the Golgi preparations, it appears that layer I is relatively acellular in nature. The GABA labeling procedure, however, shows that layer I contains several neurons although the number of these GABAergic neurons is considerably smaller than that found in all other cortical layers of the auditory cortices. Of all cortical laminae, layers II and IV seemed to have the densest accumulation of these neurons. As has been observed previously (6,30,37,48), GABAergic neurons are nonpyramidal in type. These neurons have been described as nonspinous and have the characteristics of multipolar, bitufted, and bipolar cells, which have been further characterized as basket, double bouquet, or chandelier cells on the basis of their axonal arrays (10, 30,58). With the methods employed in this study, such classifications of the GABAergic neurons were not possible but the somata are all round-to-oval and are of a relatively uniform size suggesting that they are nonpyramidal in type (Fig. 4). The relative paucity of GABAergic neurons in layer III, especially in its lower part, and in layers V and VI suggest that the majority of the neurons in these layers are probably of the pyramidal type. The distribution of GABAergic neurons has been shown to vary among other cortical areas of the monkey (25, 66). Indeed, comparison of the distribution of the GABAergic neurons of the primary auditory region (area KA) with the belt areas (i.e., area paAlt) indicates that there are relatively more GABAergic neurons in area KA than in the association cortical area paAlt (Figs. 4A and 4B). The reason for this pattern could very well be that in the primary auditory area small

OF AUDITORY

CORTEX

119

neurons are evenly distributed throughout layers II-IV, whereas in the belt areas the small neurons are more concentrated in layer IV, as seen in Nissl preparations (2% The peptidergic neurons are also of the nonspinous type and are distributed in varying degrees in specific cortical laminae of the auditory cortices. Moreover, the number of peptidergic cells varies among the subdivisions of these cortices. The ~stribution of VIP-reactive neurons in these cortical regions indicates that the primary auditory area has a limited number of these cells located mainly in layers II and III while the second auditory area has comparatively more VIP-reactive neurons distributed in layers II-V. Association areas of the STG, on the other hand, have few VIP-reactive neurons (Fig. 6A). These neurons are predominantly of the nonspinous bipolar type (Figs. 6B and 6C). The CCK-reactive neurons are sparse in the auditory cortices of the monkey and are found mainly in layers II and III (Fig. 8). These neurons appear to be nonspinous multipolar and bipolar cells (Fig. SA). SS-reactive neurons are observed in the root, belt, and core regions of the auditory-related cortices and their distributions also vary among the auditory cortical subdivisions (Fig. 7A). That is, in the primary auditory region (area KA), they are sparse and localized in layers II and III, while in the second auditory area (area proA), they are distributed in both the supra- and infragranular layers. In the association cortices (STG), the SS-reactive neurons are found again mainly in layers II and III although there are some in layers V and VI. These neurons are mainly multipolar but are occasionally bit&ted (Figs. 7B-7D). The distribution patterns and types of VIP-, CCK-, and SS-reactive neurons are thus similar to those described by others (4, 14). The SP-reactive neurons are more numerous than the VIP-, CCK-, and SS-reactive neurons. In the primary auditory region, they are found mainly in layers II and III and are localized to the medial aspect of this region (Fig. 5A). In the circular sulcus, the SP-reactive neurons are distributed in both the supra- and infragranular layers. In the association areas, the SP-reactive neurons are found mainly in layers II and III. In contrast, other studies have described these cells as being mainly in layers II, III, IV, and V of the primate visual cortex (36). These neurons are usually bipolar, although other nonspinous types are also reactive to SP antibodies (Fig. 5B and 5C). In comparison to the peptidergic neurons, the histochemically labeled cells are more numerous and have markedly different distributions patterns. The NADPH-d-positive neurons are also nonpyramidal and are distributed almost equally in the primary, secondary, and association auditory areas, and are found in both the supra- and infragranular layers (Fig. 10). In

120

CIPOLLONI

many areas, however, there are no labeled neurons in the central cortical laminae, as has been previously reported for the striate cortex of the monkey (65). The white matter underlying these cortices also contain a large number of NADPH-d-labeled neurons. In the cortex (Fig. 111, these neurons are completely labeled and are easily identified as the nonspinous bipolar, bitufted, and multipolar types seen in Golgi preparations while in the white matter, they are strictly nonspinous multipolar cells. Cytochrome oxidase positive neurons are mainly pyramidal cells (Figs. 9B and 9C) and their distribution in these cortices is limited to the association areas of STG except for a few labeled neurons in the primary auditory area (Fig. 9A). From our material it seems, therefore, that some of the neurons within the auditory-related cortices have chemical specificity and different distribution patterns that can be related to the various architectonic areas. Cortical neuropeptides have been localized in a broad class of interneurons, forming about 5-10% of the total cortical neuronal population {35). The variability of the distributions of the peptidergic neurons in the auditory cortices of the monkey has already been shown for SSreactive neurons (4). Also, in the visual cortex, SS, and neuropeptide Y plexuses are bilaminar, and in cingulate cortex their distribution is trilaminar (35). The precise functional role of these peptides in the central nervous system is not clearly established as yet but it is assumed that the peptidergic neurons have inhibitory properties because they co-localize, to some degree, with GABA (6, 36). These peptides are also thought to modulate the action of neurotransmitters (68). Although light microscopic studies have outlined the ~st~butions of the various auditory cortical afferents as well as the neurons of origin of these afferents (7, 20-22,50,53), identification of the precise neurons that receive these afferents require electron microscopic analysis. The information gained from the present study provides the necessary background for such ultrastructural studies of the connectivity of the auditory-related cortices of the monkey (e.g., 6, 8, 39).

We thank Dr. Neil Kowall and Mr. Robert Ferrante for providing some of the histo- and immunocytochemical material included in this study. We extend our sincere appreciation also to Drs. Cliiord Barnes, Asaf Keller, Edward White, and Edward Yeterian for useful comments. We are grateful to Ms. Valerie Knowlton, Mr. Bruce Ekstein, Ms. Bonnie Meek, and Ms. Dina Pandya for their technical assistance, and to Mr. Robert Landrigan for assistance with the illustrations. This study is supported by NIH grants 20967 (P.B.C.) and 16841 (D.N.P.), the Veterans A~i~stration, the Edith Nourse Rogers Memorial Veterans Hospital, Bedford, MA 01730, and the Institute for Neurological Research.

AND PANDYA

REFERENCES 1. BEAL, M. F., AND J. B. MARTIN. 1986. Neuropeptides in neurological diseases. Ann. Neural. 20: 547-565. 2. BERITOFF, J. S. 1965. Neural Mechanisms of Higher Vertebrate Behavior. Little, Brown, Boston. 3. B~I~NBERG, V., V. GuGL~~~, AND E. SADA. 1967. Correlation of crystal growth with the staining of axons by the Golgi procedure. Stain Technol. 42: 277-282. 4. CAMPBELL, M. J., D. A. LEWIS, R. BENOIT, AND J. H. MORRISON. 1987. Regional heterogeneity in the distribution of somatostatinand somatostatin-28-(1-~2)-immunoreactive profiles in monkey neocortex. J. Neurosci. 7: 1133-1144. 5. CARBONI, A. A., W. G. LAVELLE, C. L. BARNES, AND P. B. CIPOLLONI. 1990. Neurons of the lateral entorhinal cortex of the rhesus monkey: A Golgi, histochemical and immunocytochemical characterization. J. Camp. New-d 291: 583-608. 6. CIPOLLONI, P. B., AND A. KELLER. 1989. Thalam~o~ic~ synapses with identified neurons in monkey primary auditory cortex: A combined Golgi/EM and GABA/peptide immunocytochemistry study. Brain Res. 492: 347-355. 7. CIPOLLONI, P. B., AND D. N. PANDYA. 1989 Connectional analysis of the ipsilateral and contralateral afferent neurons of the superior temporal region in the rhesus monkey. J. Camp. Neurol. 281:567-585. 8. CIPOLLONI, P. B., D. N. PANDYA, AND V. M. KNOWLTON. 1986. Thalamocortieal afferents onto identified neurons of the primary auditory cortex of the rhesus monkey: An ultrastructural analysis. Sot. Neurosci. Abstr. 12: 1275. 9. CIPOLLONI, P. B., AND A. PETERS. 1983. The termination of callosal fibres in the auditory cortex of the rat: A combined Golgielectron microscope and degeneration study. J. Neurocytol. 12: 713-726. 10. DEFELIPE, J., AND A. FA~~N. 1982. A type of basket cell in superficial layers of the cat visual cortex: A Golgi-electron microscopic study. Brain Res. 224: 9-16. 11. DEFEL~PE, J., AND E. G. JONES. 1988. Histology of the visual cortex of the cat. In @jut on the Cerebral Cortex: An Annotated Trans~t~n of the Complete writhes. pp. 495-523. Oxford University Press, New York. 12. DIFIGLIA, M., G. A. GRAVELAND, AND L. SCHIFF. 1987. Cytochrome oxidase activity in the rat caudate nucleus: Light and electron microscopic observations. J. Camp. Neural. 265: 137145. 13. ELHANANY, E., AND E. L. WHITE. 1990. Intrinsic circuitry: Synapses involving the local axon collaterals of corticocortical projection neurons in the mouse primary somatosensory cortex. J. Comp. Neurol. 291: 43-54. neu14. EMSON, P. C., AND S. P. HUNT. 1984. Peptide-containing rons of the cerebral cortex. In Cerebd Cortex: Vol. 2. ~u~t~~~ Properties of Cortical Cells (E. G. Jones and A. Peters, Eds.), pp. 145-169. Plenum Press, New York. 15. FAI&N, A., J. DEFELIPE, AND J. REGIDOR. 1984. Non-pyramidal neurons: General account. In Cerebral Cortex. Vol. I. Cellular Components of the Cerebral Cortex (A. Peters and E. G. Jones, Eds.), pp. 201-253. Plenum Press, New York. 16. FELDMAN, M. L. 1984. Morphology of the neocortical pyramidal neuron. In Cerebral Cortex, Vol. 1. Cellular Components of the Cerebral Cortex (A. Peters and E. G. Jones, Eds.), pp. 123-200, Plenum Press, New York. 17. FELDMAN, M. L., AND A. PETERS. 1978. The forms of non-pyramidal neurons in the visual cortex of the rat. J. Camp. Neural. 179:761-794.

NEURONAL

ANALYSIS

18. FERRANTE, R. J., N. W. KOWALL, M. F. BEAL, E. P. RICHARDSON JR., E. D. BIRD, AND J. B. MARTIN. 1985. Selective sparing of a class of striatal neurons in Huntington’s disease. Science 230: 561-563. 19. FITZPATRICK, K. A., AND T. J. IMIG. 1978. Projections of auditory cortex upon the thalamus and midbrain in the owl monkey. J. Comp. Neural. 177: 537-556. 20. FITZPATRICK, K. A., AND T. J. IMI~,. 1980. Auditory cortico-eortical connections in the owl monkey. J. Comp. Neural. 192: 589610. 21. FITZPATRICK, K. A., AND T. J. IMIG. 1982. Organization of auditory connections. The primate auditory cortex. In Cort~al Sensory Organization: Multiple Auditory Areas. (C. N. Woolsey, Ed.), pp. 71-109. Humana Press, Clifton. 22. GALABURDA, A. M., AND D. N. PANDYA. 1983. The intrinsic architectonic and connectional organization of the superior temporal region of the rhesus monkey. J. Camp. Neural. 222: 169184. 23. GALLYAS, F., T. GORES, AND I. MERCHENTHALER. 1982. Highgrade intensification of the end-product of the diaminobenzidine reaction for peroxidase histochemistry. J. H&o&em. Cytothem. 30: 183-184. 24. GLOBUS, A., AND A. SCHEIBEL. 1967. Pattern and field in cortical structure: The rabbit. J. Comp. Neural. 131: 155-172. 25. HENDRY, S. H. C., H. D. SCHWARK, E. G. JONES, AND J. YAN. 1987. Numbers and proportions of GABA-immunoreactive neurons in different areas of monkey cerebral cortex. J. Neurosci. 7: 1503-1519. 26. HERSCH, S. M., R. J. FERRANTE, AND P. B. CIPOLLONI. 1987. Identified corticostriatal synapses involving NADPH-diaphorase positive and neuropeptide-Y immunoreactive neurons in the rhesus monkey. Sot. Neurosci. Abstr. 13: 1570. 27. HORNUNG, J. P., AND L. J. GAREY. 1981. The thalamic projection to cat visual cortex: Ultrastructure of neurons identified by Golgi impregnation on retrograde horseradish peroxidase transport. J. Neurosci. 6: 1053-1068. 28. HORTON, J. C., AND E. TESSA HEDLEY-WHITE. 1984. Mapping of cytochrome oxidase patches and ocular dominance columns in human visual cortex. Philos. Trans. R. Sot. London Ser. 3 304: 255-272. 29. HOUSER, C. R., S. H. C. HENDRY, E. G. JONES, AND J. K. VAUGHN. 1983. Morphological diversity of immunocytochemitally identified GABA neurons in the monkey sensory motor cortex.

J. Neurocytol.

12: 617-638.

30. HOUSER, C. R., J. E. VAUGHN, S. H. 6. HENDRY, E. G. JONES, AND A. PETERS. 1984. GABA neurons in the cerebral cortex. In Cerebral

31.

32.

33.

34.

Cortex.

Vol. 2. Functional

Properties

of Cortical

Cells

(E. G. Jones and A. Peters, Eds.), pp. 63-89. Plenum Press, New York. ICHIKAWA, M., K. ARISSIAN, AND H. ASANUMA. 1985. Distribution of corticocortical and thalamocortical synapses on identified motor cortical neurons in the cat: Golgi, electron microscopic and degeneration study. Brain Res. 345: 87-101. ITOH, K., A. KONISXI, S. NOMURA, N. MIZUNO, Y. NAKAMURA, AND T. SUGIMOTO. 1979. Application of coupled oxidation reaction to electron microscopic demonstration of horseradish peroxidase: cobalt-glucose oxidase method. Bruin Res. 175: 341346. Izzo, P. N., A. M. GRAYBIEL, AND J. P. BOLAM. 1987. Characterization of substance-P and (met)enkephelin-immunoreactive neurons in the caudate nucleus of cat and ferret by a single section Golgi procedure. Neuroscience 20: 577-587. JONES, E. G. 1975. Varieties and distribution of non-pyramidal

OF AUDITORY

121

CORTEX

cells in the somatic sensory cortex of the squirrel monkey. J. Comp.

Neural.

160:

205-268.

35. JONES, E. G. 1986. Neurotransmitters in the cerebral cortex. J. Neurosurg. 65: 135-153. 36. JONES, E. G., S. H. C. HENDRY, AND J. DEFELIPE. 1987. GABApeptide neurons of the primate cerebral cortex: A limited ceil class. In Cerebral Cortex. Vol. 6. Further Aspects of Cortical Function, Zncluding Hippocampus. (E. G. Jones and A. Peters, Eds.), pp. 237-266. Plenum Press, New York. 37. KELLER, A. 1989. Functional aspects of cortical circuitry. In Cortical Circuits: Synaptic Organization of the Cerebral Cortex. StrutLure, ~~~~~~, and Theory (E. L. White, Ed.), pp. 109-176. Birkhauser, Boston. 38. KELLER, A., AND E. L. WHITE. 1987. Synaptic organization of GABAergic neurons in the SmI cortex. J. Comp. Neural. 262: 1-12. 39. KNO~TON, V. M., D. A. PANDYA, B. A. EKSTEIN, AND P. B. CIPOLLONI. 1987. An ultrastructural analysis of primary auditory cortical afferents onto identified neurons of the lateral auditory association cortex of the rhesus monkey. Sot. Neurosci. Abstr.

13: 327.

40. KRNJEVIC, K. 1984. Neurotransmitters in cerebral cortex: A general account. In Cerebral Cortex. Vol. 1. Cellular Components of the Cerebral Cortex (A. Peters and E. G. Jones, Eds.), pp. 39-61. Plenum Press, New York. 41. LORENTE DE N6, R. 1922. La corteza cerebral de1 raton. I. La corteza acustica. Trab. Lab. Invest. Biol. Univ. Madrid 20: 41-78. 42. LOREN~ DE Nit, R. 1938. Cerebral cortex: Architecture, intraeortical connections, motor projections. In Physiology of the Nervous System (J. F. F&on, Ed.), pp. 288-313. 2nd ed. Oxford Univ. Press, London. 43. LUND, J. S. 1973. Organization of neurons in the visual cortex, area 17, of the monkey (Macacca mulattaf. J. Camp. Neural.

147:455-496. 44. LUND, J. S. 1984. Spiny stellate neurons. In Cerebral Cortex. Vol. 1. Cellular Components of the Cerebral Cortex (A. Peters and E. G. Jones, Eds.), pp. 255-308. Plenum Press, New York. 45. MCLEAN, I. W., AND P. K. NAKANE. 1974. Periodate-lysineparaformaldehyde: A new fixative for immunoelectron microscopy. J. Histochem. Cytochem. 22: 1077-1083. 46. MCMULLEN, N. T., AND E. M. GLASER. 1982. Morphology and laminar distribution of nonpyramidal neurons in the auditory cortex of the rabbit. J. Comp. Neural. 208: 85-106. 47. MCMULI,EN, N. T., E. M. GLASER, AND M. TAGMETS. 1984. Morphometry of spine-free nonpyramidal neurons in rabbit auditory cortex. J. Comp. Neurol. 222: 383-395. 48. MEINECKE, D. L., AND A. PETERS. 1987. GABA immunoreactive neurons in rat visual cortex. J. Comp. ZVeuroE. 261: 388-404. 49. MER~NICH, M. M., AND J. F. BRUG@. 1973. Representation of the cochlear partition on the superior temporal plane of the macaque monkey. Brain Res. 50: 275-296. 50. MESULAM, M.-M., AND D. N. PANDYA. 1973. The projections of the medial geniculate complex within the Sylvian fissure of the rhesus monkey. Brain Res. 60: 315-333. 51. MIZUKAWA, K., P. L. MCGEER, S. R. VINCENT, AND E. G. MCGEER. 1988. Ultrastructure of reduced nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase-positive neurons in the cat cerebral cortex, amygdala and caudate nucleus. Brain Res. 452:

286-292.

52. PANDYA, 11. N., M. HALLET, AND S. K. M~~~~. 1969. Intraand interhemispheric connections of the neocortical auditory system in the rhesus monkey. Brain Res. 14: 49-65.

122 53.

54.

55.

CIPOLLONI

AND

PANDYA, D. N., AND D. L. ROSENE. 1983. Termination patterns of thalamic, callosal and association a6’erents of the primary auditory area in the rhesus monkey. Soe. Neurosci. Abstr. 9: 954. PETERS, A. 1984. Chandelier cells. In Cerebral Cortex Vol. 1. CeZlular Components of the Cerebral Cortex. (A. Peters and E. G. Jones, Eds.), Plenum Press, New York. PETERS, A., AND A. FAIF&N. 1978. Smooth and sparsely spined stellate cells in the visual cortex of the rat: A study using a combined Golgi-electron microscopic technique. J. Comp. NeuroL

bra1 Cortex

71.

72.

181:129-172. 56.

57.

PETERS, A., AND E. G. JONES. 1984. Classification of cortical neurons. In Cerebral Cortex. Vol 1. Ce~u~ar ~orn~~~s of the Cerebral Cortex (A. Peters and E. G. Jones, Eds.), pp. 107-121. Plenum Press, New York. PETERS, A., C. C. PROSKAUER, M. L. FELDMAN, AND L. KIMERER. 1979. The projection of the lateral geniculate nucleus to area 17 of the rat cerebral cortex. V. Degenerating axon terminals synapsing with Golgi impregnated neurons. J. NeurocytoL

59.

Pm=, A., C. C. PROSKAUER, AND C. E. RIBAK. 1982. Chandelier cells in rat visual cortex. J. Comp. Neural. 206: 397-416. PETERS, A., AND J. REGIDOR. 1981. A reassessment of the forms of nonpyramidal neurons in area 17 of cat visual cortex. J. Comp.

60.

62. 63.

64.

74.

Cortex.

66.

67.

75. 76.

685-716.

Vol. 1. Cellular

Components

of the Cerebral

77.

Cor-

Peters and E. G. Jones, Eds.), pp. 419-445, Plenum Press, New York. POUK, J. M., AND S. VAN NOORDEN (Eds). 1986. Immunocytochemistry. Modern Methods and Applications, 2nd ed. Wright, Bristol. RAM~N Y CAJAL, S. 1899. Estudios sobre la corteza cerebral humana. Corteza visual. Rev. Trim. Microgr. 4: l-63. RAM~N Y CAJAL, S. 1955. Histologie du Systeme Nerveux de 1’Homme et des Vertebres (Translated by L. Azoulay), Instituto Ram& y Cajal, Madrid. RIBAK, C. E., J. E. VAUGHAN, AND K. SAITO. 1978. Immunoc~chemical localization of glutamic acid decarboxylase in neuronal somata following colchicine inhibition of axonal transport. BrainRes.

65.

203:

PEERS, A., AND R. L. SAINT MARIE. 1984. Smooth and sparsely spinous nonpyramidal cells forming local axonal plexuses. In Cerebral tex (A.

61.

Neural.

140:315-332.

SANDELL, J. H. 1986. NADPH diaphorase histochemistry in the macaque striate cortex. J. Comp. Neurol. 261: 388-397. SCHWARTZ, M. L., D.-S. ZHENG, AND P. S. GOLDMAN-RAKIC. 1988. Periodicity of GABA-containing cells in primate prefrontal cortex. J. Neurosci. 8: 1962-1970. SELDON, H. L. 1981. Structure of human auditory cortex. I. Cytoarchitectonics and dendritic distributions. Brain Res. 229:

69.

70.

of the Cerebral

Cortex

(A.

79.

181:62'7-662.

WHITE, E. L. 1979. Thalam~o~ic~ synaptic relations: A review with emphasis on the projection of specific thalamic nuclei to the primary sensory areas of the neocortex. Brain Res. Rev. 1: 275-311. WHITE, E. L. 1989. Cell types. In Cortical Circuits. Synaptic Organization

of the Cerebral

Cortex.

Structure,

Function,

and Theory

(E. L. White, Ed.), pp. 19-45. Birkhiiuser, Boston. WHITE, E. L. 1989. Synaptic connections between identified ele80. ments. In Cortical Circuits: Synaptic Organization of the Cerebral Cortex. Str~t~re, Function, and Theory (E, L. White, Ed.), pp. 46-105. Birkhiiuser, Boston. 81. WHITE, E. L., AND S. M. HERSCH. 1981. Thalamocortical synapses of pyramidal cells which project from SmI to MsI cortex in the mouse. J. Comp. Neural. 198: 167-181. 82. WHITE, E. L., S. M. HERSCH, AND M. P. ROCK. 1980. Synaptic sequences in mouse SmI cortex involving pyramidal cells labeled by retrograde filling with horseradish peroxidase. Neurosci. Lett. 19: 149-154. 83. WHITE, E. L., AND M. P. ROCK. 1980. Three-~mension~ aspects and synaptic relationships of a Golf-imp~~ated spiny stellate cell reconstructed from serial thin sections. J. Neurocytol. 9:

615-636.

SIGGINS, G. R., AND D. L. GRUOL. 1986. Mechanisms of transmitter action in the vertebrate central nervous system. In Handbook of Physiology (V. B. Mountcastle and S. R. Geiger, Eds.), pp. l-114. American Physiological Society, Bethesda. SILLITO, A. M. 1984. Functional considerations of the operation of GABAergic inhibitory processes in the visual cortex. In Cere-

brat Cortex.

Components

T~MB~L, T. 1978. Some Golgi data on visual cortex of the rhesus monkey. Acta. Morphol. Acad. Sci. Hungary 26: 116-138. VALVEF~DE, F. 1970. The Golgi method. A tool for comparative structural analyses. In Contemporary Methods in Neuroanatomy (W. J. H. Nauta and S. 0. E. Ebbesson, Eda.). Springer-Verlag, New York. VALVERDE, F. 1971. Short axon neuronal subsystems in the visual cortex of the monkey. Int. J. Neurosci. 1: 181-197. VALVERDE, F. 1978. The organization of area 18 in the monkey: A Golgi study. Anat. Embryol. 151: 305-334. 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.

78.

277-294. 68.

Vol. 1. Cellular

Peters and E. G. Jones, Eds.), pp. 337-360. Plenum Press, New York. SOMOGYI, P., T. F. FREUND, J. Y. WV, AND A. D. SMITH.1983. The section-Golgi 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 9: 475-490. SOMOGYI, P., A. J. HODGSON, AND A. D. SMITH. 1979. An approach to tracing neuron networks in the cerebral cortex and basal ganglia. Combination of Golgi staining, retrograde transport of horseradish peroxidase and anterograde degeneration of synaptic boutons in the same material. J. Neurosci. 4: 18051855.

73.

8:331-357. 58.

PANDYA

Vol. 2. Fu~tio~~

Properties

of Cortical

Cells (E. G.

Jones and A. Peters, Eds.), pp. 91-117. Plenum Press, New York. SOMOGYI, P. AND A. COWEY. 1984. Double bouquet cells. In Cere-

84.

WINER, J. A. 1984. The non-pyramidal cells of layer III in primary auditory cortex (AI) of the cat. J. Comp. Neural. 229: 512530.

85.

86.

WONG-RILEY, M. 1979. Changes in the visual system of monocularly sutured or enucleated cats demonstrable with cytochrome oxidase hist~hemistry. Bruin Res. 171: 11-28. WONG-RILEY, M. T., AND E. W. CARROLL. 1984. ~antitative light and electron microscopic analysis of cytochrome oxidaserich zones in V II perstriate cortex of the squirrel monkey. J. Comp.

Neural.

222:

18-37.