the midbrain periaqueductal gray in the rat. ii. a golgi analysis

16
THE JOURNAL OF COMPARATIVE NEUROLOGY 237:460-475 (1985) The Midbrain Periaqueductal Gray in the Rat. 11. A Golgi Analysis ALVIN J. BEITZ AND R. DAVID SHEPARD Department of Veterinary Biology, University of Minnesota, St. Paul, Minnesota 55108 ABSTRACT This study consists of a detailed analysis of neurons in the midbrain periaqueductal gray of the rat utilizing four variants of the Golgi technique. Neurons were classified into three major categories based on soma shape, number of primary dendrites, number of dendritic bifurcations, interspinous distance, axonal origin, and axon trajectory. Neurons in each category were further subdivided into large and small varieties based predominantly on soma size and dendritic patterns. Both quantitative and qualitative data concerning each neuronal type is provided as well as data relating to its relative distribution among the four periaqueductal gray subdivisions. The small bipolar neuron, characterized by its small size and spindle-shaped soma, was the most prominent cell type observed, composing 37% of the impregnated neurons in our material. This cell type was most numerous in the medial subdivision and least prominent in the dorsolateral subdivision. The small triangular neuron composed 23% of the neuronal population and was relatively evenly distributed through the periaqueductal gray. The remaining four cell types include the large and small multipolar neurons, the large fusiform neurons, and the large triangular neurons. Axons origi- nated from either the perikaryon or a proximal dendrite, with a dendritic origin being most common for large and small triangular neurons and large fusiform neurons. The trajectory of axons in single thick coronal sections originating from periaqueductal gray neurons is typically away from the mesencephalic aqueduct. The exact trajectory is dependent on the location of the neuron. Axons arising from cells in the dorsal subdivision usually project in a dorsal or dorsolateral direction while axons of ventrolateral neurons may project dorsally, laterally, or ventrally. In sum, these data indicate a complex level of internal organization of the periaqueductal gray. The results are discussed in terms of previous immunohistochemical studies of neurons in this region. Key words: neuronal types, analgesia, PAG subdivisions The midbrain periaqueductal gray (PAG) is a functionally complex region which receives extensive input from all levels of the central nervous system (Beitz, 82a; Liu, '83; Mantyh, '82b; Marchand and Hagino, '83; Morrell et al., '81). These numerous sources of afferent projections allow the PAG to be influenced by motor, sensory, limbic, and autonomic structures (Beitz, '82a). Many areas such as the hypothalamus, subthalamus, reticular formation, raphe nuclei, locus ceruleus, and spinal cord which project to the central gray also receive reciprocal connections back from this midbrain region (Beitz et al., '83a; Mantyh, '83a,b; Ruda, '76). This diverse interconnectivity probably under- lies the numerous functional roles attributed to the PAG. This region has been implicated in central analgesic mech- anisms (Basbaum and Fields, '84; Gerhart et al., '84; Willis et al, '84), vocalization (Jurgens and Pratt, '79), control of reproductive behavior (Sakuma and Pfaff, '79), aggressive behavior (Mos et al., '82), control of upward gaze (Thames et al., '84), and various autonomic functions (Skultety, '59). Although numerous reports have appeared regarding the connections and functions of the PAG, data concerning the morphology of this region, especially in the rat, are scarce. Aside from the work of Mantyh ('82a) we are not aware of any other investigations of the rat central gray which have employed the Golgi technique. The following study was designed to analyze the morphology and cytoarchitecture of the rat PAG using several variations of the Golgi technique. In addition the cell types which compose this region are Accepted February 6,1985. 0 1985 ALAN R. LISS, INC.

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Page 1: The midbrain periaqueductal gray in the rat. II. A Golgi analysis

THE JOURNAL OF COMPARATIVE NEUROLOGY 237:460-475 (1985)

The Midbrain Periaqueductal Gray in the Rat. 11. A Golgi Analysis

ALVIN J. BEITZ AND R. DAVID SHEPARD Department of Veterinary Biology, University of Minnesota, St. Paul, Minnesota 55108

ABSTRACT This study consists of a detailed analysis of neurons in the midbrain

periaqueductal gray of the rat utilizing four variants of the Golgi technique. Neurons were classified into three major categories based on soma shape, number of primary dendrites, number of dendritic bifurcations, interspinous distance, axonal origin, and axon trajectory. Neurons in each category were further subdivided into large and small varieties based predominantly on soma size and dendritic patterns. Both quantitative and qualitative data concerning each neuronal type is provided as well as data relating to its relative distribution among the four periaqueductal gray subdivisions. The small bipolar neuron, characterized by its small size and spindle-shaped soma, was the most prominent cell type observed, composing 37% of the impregnated neurons in our material. This cell type was most numerous in the medial subdivision and least prominent in the dorsolateral subdivision. The small triangular neuron composed 23% of the neuronal population and was relatively evenly distributed through the periaqueductal gray. The remaining four cell types include the large and small multipolar neurons, the large fusiform neurons, and the large triangular neurons. Axons origi- nated from either the perikaryon or a proximal dendrite, with a dendritic origin being most common for large and small triangular neurons and large fusiform neurons. The trajectory of axons in single thick coronal sections originating from periaqueductal gray neurons is typically away from the mesencephalic aqueduct. The exact trajectory is dependent on the location of the neuron. Axons arising from cells in the dorsal subdivision usually project in a dorsal or dorsolateral direction while axons of ventrolateral neurons may project dorsally, laterally, or ventrally. In sum, these data indicate a complex level of internal organization of the periaqueductal gray. The results are discussed in terms of previous immunohistochemical studies of neurons in this region.

Key words: neuronal types, analgesia, PAG subdivisions

The midbrain periaqueductal gray (PAG) is a functionally complex region which receives extensive input from all levels of the central nervous system (Beitz, 82a; Liu, '83; Mantyh, '82b; Marchand and Hagino, '83; Morrell et al., '81). These numerous sources of afferent projections allow the PAG to be influenced by motor, sensory, limbic, and autonomic structures (Beitz, '82a). Many areas such as the hypothalamus, subthalamus, reticular formation, raphe nuclei, locus ceruleus, and spinal cord which project to the central gray also receive reciprocal connections back from this midbrain region (Beitz et al., '83a; Mantyh, '83a,b; Ruda, '76). This diverse interconnectivity probably under- lies the numerous functional roles attributed to the PAG. This region has been implicated in central analgesic mech- anisms (Basbaum and Fields, '84; Gerhart et al., '84; Willis

et al, '84), vocalization (Jurgens and Pratt, '79), control of reproductive behavior (Sakuma and Pfaff, '79), aggressive behavior (Mos et al., '82), control of upward gaze (Thames et al., '84), and various autonomic functions (Skultety, '59).

Although numerous reports have appeared regarding the connections and functions of the PAG, data concerning the morphology of this region, especially in the rat, are scarce. Aside from the work of Mantyh ('82a) we are not aware of any other investigations of the rat central gray which have employed the Golgi technique. The following study was designed to analyze the morphology and cytoarchitecture of the rat PAG using several variations of the Golgi technique. In addition the cell types which compose this region are

Accepted February 6,1985.

0 1985 ALAN R. LISS, INC.

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GOLGI ANALYSIS OF RODENT PAG

described as well as their distribution among the four PAG subdivisions. The results are compared with the data ob- tained by Mantyh ('82a) in the rat, cat, and monkey and with the data presented by Liu and Hamilton ('80) and Tredici et al. ('83) for the feline central gray.

461

drites; (3) number of dendrite bifurcations; (4) interspinous distance; (5) axonal origin; and (6) axon trajectory. Soma size and interspinous distance were measured directly from the microscope using an ocular grid as well as from camera lucida drawings of PAG neurons.

RESULTS A number of different types of Golgi methodologies were

used to impregnate cells of the periaqueductal gray. The number of neurons in the PAG with impregnated dendritic trees ranged from a minimum of zero to a maximum of about 500. Axons were examined originating from PAG neurons; in other material only axons were impregnated and their origin could not be determined. In additicn to neurons glial cells were also impregnated.

Neuronal classification Neurons were classified into three major classes: fusi-

form, triangular, and stellate. Each of these three cell types was further subdivided into small and large varieties based on size and dendritic characteristics. The major character- istics of each neuron type are described in detail below.

Fusiform neumm. The fusiform neurons were charac- terized by their elongated perikarya and the bipolar ar- rangement of their processes. This neuron type was the most prominent variety encountered in the PAG, making up 48.4% of the impregnated neurons studied (Table 1). The small fusiform cell, termed the small bioplar neuron, com- posed 36.8% of the neuron population and displayed a spin- dle-shaped perikaryon which ranged in length from 13 to 20 pm (Table 2, Figs. 1, 8). One or two primary dendrites were observed to arise from each pole of the perikaryon (Fig. 1). Occasionally small bipolar neurons were encoun- tered in which a single dendrite issued from one pole of the perikaryon and an axon arose from the opposite pole. In 60% of the small bipolar neurons studied, however, the axon arose from a primary or secondary dendrite (Table 2). Dendrites of this cell type typically branched two to three times, creating a rather narrow oblong dendrite field. Den- dritic spines were prominent, being found every 4-10 pm along the dendritic tree.

Large fusiform neurons displayed perikarya which ranged in size from 22 to 28 pm and gave rise to two to three primary dendrites (Figs. 1, 8). Two varieties of the large fusiform cells were evident in our material. The first sub- type displayed a narrow dendritic field ranging from 50 to 140 pm in diameter and prominent dendritic spines (occur-

METHODS Serial sections of midbrains from six young (8-24 days

postnatal) and 30 adult (225-275 g) male, Sprague-Dawley rats were made after application of four variants of the Golgi method the Golgi-Kopsch technique (Colonnier, '64), a chlorate formaldehyde modification of the Golgi method (Ramon Moliner, '57), the triple impregnation osmium di- chromate method (Palay and Chan-Palay, '741, and the Braitenburg technique (Braitenburg et al., '67). The first three procedures were used successfully in our laboratory in a previous study of the medial cerebellar nucleus in the rat (Beitz and Chan-Palay, '79). The brains were embedded in parlodion and sectioned at 200 pm on a sliding micro- tome in the coronal, horizontal, or sagittal plane. Brain- stems in which the periaqueductal gray were well impregnated were examined. Four impregnated brainstems were counterstained with cresyl violet to ascertain the pre- cise location of the nuclear boundaries of the PAG and to delimit the boundaries of intrinsic nuclei, such as the dorsal raphe and dorsal tegmental nuclei.

A Leitz microscope equipped with a camera lucida attach- ment was used to trace dendritic arborizations and axonal ramifications. Drawings were done with a 100 x Leitz Flu- otar (NA = 1.3), 63 x Zeiss Planapo (NA = 1.4) = 50 x Wild oil objective (NA = 1.0). All had long working distances suitable for thick Golgi sections.

In order to carefully study the organization of neurons and their processes in the PAG, two brains with extremely fine impregnation were chosen and all impregnated neu- rons in each section through the PAG from each brain were drawn with camera lucida. The margin of the mesence- phalic aqueduct and the peripheral border of the PAG were drawn to scale with the aid of a wall projector. These two sets of drawings of all PAG neurons allowed evaluation of neuronal relationships within the PAG as a whole and within each of the four proposed PAG subdivisions de- scribed in an accompanying paper (Beitz, '85).

Neuron classification Neurons were categorized based on the following six cri-

teria: (1) soma size and shape; (2) number of primary den-

TABLE 1. Percentage of Each Neuron Type in the Periaqueductal Gray and Within Each of the Four Subdivisions

Entire Ventrolateral Dorsolateral Dorsal Medial PAG subdivision subdivision subdivision subdivision (Yo) (70) (%I (%) (%I

Small bipolar 36.8 32.8 11.1 40.6 62.2

Large fusiform 11.6 14.3 11.1 19.2 2.7

Small triangular 23.2 20.0 26.9 19.2 24.3

neuron

neuron

neuron Large triangular 10.0 10.0 15.8 11.0 0

neuron Small multipolar 10.5 14.3 7.9 5.2

neuron Large multipolar 7.9 8.6 12.7 4.8

14.8

0

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462 A.J. BEITZ AND R.D. SHEPARD

TABLE 2. Quantitative Data for Each of the Six Neuronal Types Identified in the PAG

Soma Dendritic Interspinous Size Primary bifurcations distance Axonal Axon

Neuron type Olm) dendrites (Noldendrite) (pm) origin trajectory

Small bipolar 13-20 1-3 2-3 neuron

Large fusiform neuron

22-38 2-4

Small triangular 14-18 3-4 neuron

Large triangular 19-28 3-4 neuron

2-3

1-3

2-4

Small multipolar 12-20 3-5 2-4 neuron

Soma 40% Lateral 40% Primary 46.7% Dorsolateral 33%

Ventrolateral 13.5% Secondary 13.3% Ventromedial 13.5%

4-10 dendrite

dendrite

4-7 or

18-22

6-10

8-18

Primary 83.4% dendrite 83.4% Ventrolateral 38.5%

Secondary 16.6% Dorsal 30.75% dendrite Lateral 30.75%

Lateral 36.3% Soma 30.7% Ventrolateral 27.3% Primary 69.3% Dorsal 22.270

dendrite Dorsomedial 14.2%

Soma 11.0% Primary 55.7% Lateral 75%

dendrite Ventral 25%

dendrite Secondary 33.3%

Lateral 57.14 3-10 Soma 47.5% Dorsal 14.3%

Primary 52.5% Dorsolateral 14.3% dendrite Ventral 14.3%

Large Soma 71.4% Lateral 40% multipollar 22-32 4-8 2-4 7-15 Primary 28.6% Dorsal 40%

dendrite Ventromedial 20% neuron

ring every 7-15 pm). The second subtype exhibited an ex- tensive dendritic arborization which created a wide dendritic field ranging from 140 to 310 pm in diameter. This neuron variety had an ovoid-to pyramid-shaped peri- karyon and displayed few dendritic spines. Spines were limited to distal dendrites and occurred every 18-24 pm.

Rungular-shaped neurons. The second major class of PAG neurons was named after the shape of their perikarya and was divisible into large and small varieties. Small triangular neurons composed 23.2% of the PAG neuronal population and their neuronal length varied from 14 to 18 pm (Tables 1,2). Three to four primary dendrites arose from the soma and branched one to three times to form an ellip- soidal dendritic territory ranging in diameter from 70 to 130 pm (Figs. 2, 9). Spines occurred every 6-10 pm along the dendritic tree.

Large triangular neurons made up 10% of the PAG neu- ronal population and had soma lengths of 19-28 pm (Figs. 2,9). Primary dendrites of these cells branched two to four times to form dendritic fields that varied from an ellipsoidal to almost spherical shape. The dendritic field measured 120-200 pm in diameter and was larger than that of the small triangular neurons. Dendritic spines occurred every 8-18 pm. The axon of this cell type typically arose from a primary or secondary dendrite rather than the soma and traveled away from the mesencephalic aqueduct to enter the mesencephalic reticular formation or the overlying colliculi.

Multipolar neurons. The stellate or multipolar neurons were polygonal to spherical in shape and composed 18.4% of the PAG neurons impregnated. Small multipolar neurons were more prominent than large multipolar neurons and gave rise to three to five primary dendrites (Tables 1, 2). Large multipolar neurons exhibited perikarya measuring

22-32 pm in length from which four to seven primary dendrites arose (Figs. 3, 10). Dendritic spines were absent on the proximal dendrites of the large multipolar neurons but occurred every 7-15 pm on the distal dendrites. Dendri- tic spines were typically observed every 3-10 pm along the dendritic tree of the small multipolar neurons. Occasionally a small multipolar neuron was encountered which was devoid of dendritic spines. The axons of small multipolar neurons arose from the primary dendrite in 52.5% of the cells sampled while the axons of the large multipolar neu- rons arose form the soma in 71.43% of these cells.

PAG subdivisions The organization of the neuron types within each of the

four PAG subdivisions will be described. The four subdivi- sions which make up the rat PAG are described in an accompanying paper (Beitz, '85).

Abbreviations

Aq Mesencephalic aqueduct ax Axon DLS Dorsolateral subdivision DRN Dorsal raphe nucleus DS Dorsal subdivision LFN Large fusiform neuron LMN Large multipolar neuron LTN Large triangular neuron MS Medial Subdivision SBN Small bipolar neuron SMN Small multipolar neuron STN Small triangular neuron VLS Ventrolateral subdivision

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Fig. 1. Camera lucida drawing of two small bipolar neurons (SBN and SBN-2) and one large fusiform neuron from the midbrain PAG of the rat. The exact location and orientation of each neuron in the PAG is shown on the inset. The axon (ax) of the SBN located laterally arises from a primary dendrite and travels in a lateral direction to exit the PAG. SBN-2 is located

in the medial subdivision and gives rise to an axon that travels dorsally, giving off a collateral which is directed laterally. Both SBNs display numer- ous dendritic spines (see also Fig. 8). The LFN has fewer spines and gives rise to an axon which is also directed in a lateral direction.

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464 A.J. BEITZ AND R.D. SHEPARD

i i

Fig. 2. Camera lucida drawing of a small triangular neuron (STN) from the medial subdivision and a large triangular neuron (LTN) from the dorsolateral subdivision. The axon of the STN arises from a primary dendrite while the axon of the LTN arises form the cell soma. Note the elongated spines on the dendrites of the LTN.

Medial subdivision. The medial subdivision surrounded the mesencephalic aqueduct and contained four of the six neuron types (Table 1). Small bipolar neurons were the most prominent cell type in this subdivision, comprising 62.2% of the neuron population. Dendrites of small bipolar neurons in the medial subdivision were oriented predomi- nantly parallel to the wall of the cerebral aqueduct (Fig. 4). Axons from these neurons traveled in a lateral or dorsolat- era1 direction to enter the ventrolateral or dorsolateral sub- divisions. Small triangular and small multipolar neurons represented 39.1% of the neuron population. The dendrites of small triangular neurons also coursed parallel to the wall of the mesencephalic aqueduct, while those of the small multipolar cells were randomly oriented. Large fusi- form neurons were the only large cell variety located in the medial subdivision. These neurons were usually encoun- tered in the dorsal half of the medial subdivision and gave rise to axons that traveled dorsally or dorsolaterally to enter the dorsolateral PAG subdivision or the overlying colliculi.

L)orsal subdivision. All six neuron types were present in this subdivision although the small bipolar, large fusiform, and small triangular neurons were the most prominent

(Table 1). The dendrites of small bipolar neurons were aligned in either a dorsal to ventral or a medial to lateral orientation (Fig. 5). The remaining neuron types displayed dendrites oriented randomly. Most neurons encountered had one or more dendrites that extended into the underly- ing medial subdivision or the overlying colliculi. Axons of the small bipolar neurons could be followed laterally into the dorsolateral PAG subdivision or dorsally toward the overlying colliculi. Axons of large fusiform, small triangu- lar, and large triangular neurons traveled predominantly dorsally and could often be observed to enter the overlying colliculi or collicular commissures. Axons of the multipolar neurons in this subdivision could not be traced.

Dorsolateral subdivision. The small triangular and bi- polar neurons were the most prominent neurons in this subdivision (Fig. 6). The dendrites of these cells were often arranged in a mediolateral direction so that they displayed a tangential or radial orientation to the wall of the mesen- cephalic aqueduct. The axons of the small triangular neu- rons coursed in a lateral or dorsomedial direction while those of the small bipolar neurons traveled ventrally or ventrolaterally. The large triangular neuron was more prominent in this subdivision than in the other three (Table

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GOLGI ANALYSIS OF RODENT PAG 465

Fig. 3. Camera lucida drawing of a small multipolar neuron (SMN) from the dorsal subdivision and a large multipolar neuron (LMN) from the dor- solateral subdivision. The location and orientation of the neurons in the

PAG are shown on the inset. The dendrites of the LMN exhibit numerous spines while no spines were evident on the SMN.

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466

DLS

VLS

A.J. BEITZ AND R.D. SHEPAW

i

i

Fig. 4. Camera lucida drawing of all the neurons impregnated with the Braitenburg Golgi method in the medial subdivision of the PAG from four consecutive sections through the rat midbrain. Two SBNs and STNs are indicated as well as one SMN. The cells have been coded to show their relative depth in the neuropil, the cells rendered black being most superti- cia1 and those lightly stippled deepest. Both the VLS and DLS as well as the mesencephalic aqueduct (Aq) are shown.

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GOLGI ANALYSIS OF RODENT PAG 467

DLS

Fig. 5. The cytology and organization of neurons in the dorsal subdivision of the PAG. Two STNs, an LTN, and an SMN are indicated. Several neurons in this division can be seen to be oriented vertically.

1). The long axis of the elliptical dendritic field of this cell was oriented in a mediolateral direction in most cases but occasionally was directed dorsoventrally. Axons of the large triangular neurons were always directed laterally. The ax- om of large multipolar neurons also traveled laterally and could be followed into the reticular formation. The axons of some large multipolar neurons were directed ventromedi- ally and occasionally a dorsally directed axon was observed.

Venhlateml subdiuision. Small bipolar and triangular neurons represented 52.8% of the neuronal population of this subdivision (Table 1, Fig. 7). The dendritic trees of the majority of small bipolar neurons were oriented from dor- solateral to ventromedial, while the long axes of the dendri- tic fields of the small triangular neurons were typically oriented in a dorsomedial to ventrolateral direction. The majority of axons of both the small bipolar and small tri-

angular neurons were directed laterally or ventrolaterally toward the reticular formation. Occasionally ventromedi- ally or dorsomedially directed axons were encountered. The axons of the large fusiform and large multipolar neurons traveled laterally into the reticular formation or traversed the ventrolateral subdivision to enter the dorsolateral sub- division. The second subtype of large fusiform neurons with a pyriform-shaped perikarya and few dendritic spines was the most prominent in this subdivision. These neurons typ- ically had dendritic fields oriented in a dorsolateral to ven- tromedial or lateral to medial direction. Finally, the axons of large triangular and small multipolar neurons in the ventrolateral subdivision could be followed laterally away from the mesencephalic aqueduct. The axons of small mul- tipolar neurons could also be traced dorsally into the dor- solateral subdivision.

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468

Y

A.J. BEITZ AND R.D. SHEPARD

'. -. '.

'..\

Fig. 6. Camera lucida drawing of all the neurons impregnated with the Braitenburg Golgi method in the dorsolateral subdivision from four consecutive sections through the rat midbrain. The MS and VLS are indicated.

DISCUSSION In the present study we have attempted to examine in

detail the anatomical organization of the midbrain peria- queductal gray. Our results will first be compared with previous Golgi studies of this region and then discussed in terms of immunohistochemical data related to PAG neu- rons. Finally, the functional organization of the PAG will be reviewed.

Neuronal classification A review of the literature indicates that there are as

many schemes for classifying PAG neurons as there are investigators who have attempted to classify the nerve cells of this region. Some workers have used the size and shape of the perikaryon and the number of processes as a basis for defining neuronal types (Liu and Hamilton, '80; Man- tyh, '82a) while others (Tredici et al., '83) have used quan- titative data on dendritic bifurcations, interspinous distance, and axonal size as a basis for their neuronal types. Moreover, Laemle ('79) has defined three major cell groups in the lateral PAG based upon cellular configuration and orientation. Although each of these classification schemes

has its own merit, some tend to be cumbersome because of the large diversity of neuronal varieties described while others are of little practical use to immunohistochemists or investigators using retrograde labeling procedures because these techniques usually do not label distal dendrites or dendritic spines.

The classification system proposed here is based on the original scheme provided by Ramon y Cajal ('ll), who stud- ied the neuronal structure of the PAG and described fusi- form, triangular-shaped, and stellate cells within this region. In the present study these three cell types are veri- fied and further subdivided into large and small varieties. In addition quantitative data concerning the axons and dendrites of each cell type are provided. Although a few neurons were encountered which could not be easily cate- gorized into one of these three neuronal groups, the major- ity of cells were readily classified. We believe that this classification scheme represents a reliable, workable sys- tem that can be easily utilized by other investigators whose work includes the analysis of PAG neurons.

A comparison of the fusiform neurons identified in the present study with those described by other investigators suggests that this neuron type is comparable to the type I

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GOLGI ANALYSIS OF RODENT PAG 469

Fig. 7. The cytology and organization of neurons in the ventrolateral subdivision of the PAG. Examples of LFNs, SBNs, STNs, and an SMN are shown.

neuron of Liu and Hamilton ('801, the type 4 neuron of Tredici et al. ('831, the fusiform and pyramidal neurons of Mantyh ('82a), and the vertical cells described by Laemle ('79). Tredici et al. ('83) indicated that their type 4 cells compose 13% of the neuronal population, while Liu and Hamilton ('80) reported an 18% frequency of their type I neuron. Laemle ('79) reported that the vertical cells repre- sented 40-45% of the total cell population of the lateral PAG in her material. This is comparable to the 41.9% aver- age frequency of both large and small fusiform neurons in the lateral PAG determined from our Golgi preparations.

There are several explanations that could account for discrepancies in the frequencies of particular neuron types reported in the various studies. One obvious explanation is the unpredictable nature of the Golgi procedure. Local im- pregnation tendencies vary with the age and species of the animal, and appear to depend on many unknown and there- fore uncontrollable factors (Valverde, '70). The studies of

Liu and Hamilton ('80), Mantyh ('82a), and Tredici et al. ('83) are limited to the extent that only one Golgi procedure was used to impregnate PAG neurons. To circumvent some of the problems engendered by the practical difficulties of the method itself, it is best to use several variants of the G d g i procedure and to examine large numbers of Golgi preparations through the region of interest. In this way, it is hoped that most cell types will be encountered and ade- quately sampled. In the present study, four variations of the Golgi technique, including Golgi Kopsch and rapid Golgi methods, were used. Neuronal processes, especially axons, were best visualized with the rapid Golgi and Golgi Kopsch procedures. Large PAG neurons were best impregnated with the Braitenburg technique ('67) whereas the small neurons showed fine delicate impregnation in both Golgi Kopsch- and Braitenburg-stained material.

The triangular cell type was reported to compose 30% of total impregnated cell population in the study of Liu and

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Figure 8

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GOLGI ANALYSIS OF RODENT PAG 471

Hamilton (‘80). “his is in good agreement with the 33.2% frequency observed in our material. The triangular cell is included as part of the type I cell category of Tredici et al. (‘83) and as part of the multipolar cell category of Mantyh (‘82a). Multipolar neurons identified in the present investi- gation are comparable to the type I11 b, c, and d neurons of Liu and Hamilton (‘80), the type 2 and 3 cells and part of the type 1 neurons of Tredici et al. (‘831, and the stellate cell and part of the multipolar cell categories of Mantyh (‘82a). The multipolar variety was unevenly distributed in our material, composing 22.9% and 20.6% of the impreg- nated neurons of the ventrolateral and dorsolateral subdi- visions, respectively, but only 10% of the neuronal population of the dorsal subdivision. Liu and Hamilton (‘80) also found an abundance of type TI1 cells in the lateral PAG subdivisions.

Immunohistochemical analysis of cell types Immunohistochemical studies of neuropeptide and neu-

rotransmitter localization in the PAG suggest that individ- ual substances are contained in several neuronal types rather than being localized to only one cell variety. A recent study of enkephalin immunoreactive cells in the PAG indi- cates that this pentapeptide is localized to multipolar, tri- angular, and fusiform neurons (Moss et al., ’83). It is interesting that enkephalin immunoreactivity in the lat- eral PAG was found predominantly in small bipolar neu- rons while labeled triangular and multipolar neurons were most prevalent in the dorsal PAG. Substance P immuno- reactive neurons in the PAG appear to be predominantly of the small bipolar variety, although large and small trian- gular cells are also labeled (Beitz, ’82b; Moss and Basbaum, ’83). Neurotensin immunoreactivity is most prevalent in small bipolar and small and large multipolar neurons (Beitz et al., ’83b). Cholecystokinin octapeptide occurs in small bipolar, large fusiform, small multipolar, and small trian- gular PAG neurons, while serotonin is prominent in large multipolar, small triangular, and small bipolar neurons (Beitz et al., ’83b).

The fact that there is a limited number of cell types present in the PAG together with the data indicating that each type is associated with several neuropeptides and neu- rotransmitters suggest that cell morphology is a poor pre- dictor of cell function in the PAG. This idea receives support from a recent intracellular electrophysiological analysis of PAG neurons by Reichling et al. (‘84) in which cells were characterized with natural and electrical stimuli, labelled with horseradish peroxidase, and reconstructed. These in- vestigators found no correlation between the physiological properties and the morphological characteristics of the neu- rons sampled. It should be noted, however, that this study only focused on PAG neurons responsive to nociceptive stimuli and it does not, therefore, rule out a possible corre-

Fig. 8. Photomicrographs of Golgi-impregnated SBNs and LFNs from the rat PAG. A. Low-power photomicrograph ( X 130) illustrating two SBNs (1) and one LFN (4) in the dorsolateral and dorsal portions of the PAG. Two SMNs (2) and one STN (3) are also illustrated. B. SBN from the medial subdivision (MS). Note that the orientation of the cell parallels the wall of the mesencephalic aqueduct (As). This is typical of neurons in the MS. The axon (arrow) is seen to arise from a dendrite. ~ 2 8 0 . C. A low-magnification photomicrograph ~ 2 4 0 of a Golgi-impregnated section that has been coun- terstained with cresyl violet to aid in the delineation of nuclear boundaries. An LFN (straight arrow) is shown in the lateralmost aspect of the VLS. Two neurons of the mesencephalic nucleus of V are indicated by curved arrows. D. A high-magnification photomicrograph ( ~ 5 2 0 ) of an SBN illustrating denritic spines (straight arrow) and the axon (curved arrow) arising from a primary dendrite. E. A photomontage of a LFN exhibiting a pyramidal shape. The axon (arrow) is also seen to arise from a primary dendrite.

Fig. 9. A. Photomicrograph of a STN in the medial subdivision of the PAG. X280. B. Two LTNs from the ventrolateral subdivision are illustrated. An axon (arrow) can be seen arising from the soma of the LTN at the top of the picture, while it arises from a primary dendrite of the LTN in the lower part of the field. ~ 3 2 0 .

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Fig. 10. A. A photomontage of a small multipolar neuron located in the dorsal subdivision of the PAG. A STN which is slightly out of focus can be observed a t the bottom of the field. The axon (large arrow) of the SMN originates from the cell soma. Occasional dendritic spines are also evident (small arrows). x650. B. Photomicrograph of a LMN from the dorsolateral subdivision. A LTN and a SBN are also evident. ~ 2 8 0 .

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GOLGI ANALYSIS OF RODENT PAG 473

lation of cell morphology with the numerous other proposed parts of the PAG in the rat it is necessary to apply intensi- functions of the PAG which are described below. ties of central stimulation that also trigger other marked

behavioral responses. Genuine analgesic effects could only Functional implications be produced by stimulation of limited zones in the ventral In an accompanying paper (Beitz, '85) the existence of PAG. These studies provide support for the existence of

four subdivisions in the rat PAG is described and in the dorsal and ventrolateral PAG subdivisions and further sug- present study we have depicted the neuronal types associ- gest that the analgesia induced by ventral stimulation is ated with each division. In the final portion of this discus- dependent on a separate descending pathway from SPA sion we will attempt to correlate specific functions with elicited in dorsal areas. particular PAG subdivisions. Although the PAG has been Evidence which supports the existence of a functional implicated in several functions, its role in brainstem anal- dorsolateral subdivision can be found in studies of the lor- gesic mechanisms has received the focus of attention in dosis reflex, vocalization, and escape responses. Lesions of recent years. Analgesia can be generated from all regions the dorsal portion of the PAG have been shown to produce of the PAG, but several workers have reported that the an immediate decline in performance of the lordosis reflex ventrolateral region is the most effective (Dennis et al., '80; in estrogen-primed ovariectomized female rats (Sakuma and Fardin et al., '84b; Gebhart and Toleikis, '78; Mayer and PfafT, '79). More recently, Sakuma and PfafT ('83) have Liebeskind, '74). Morphine microinjected into the ventral shown that infusion of luteinizing hormone-releasing hor- PAG, for example, is more effective in producing antinoci- mone (LHRH) into the dorsolateral PAG facilitates lordosis ception in the tail flick and hot plate tests than dorsal PAG while infusion of an anti-LHRH globulin into the PAG injections (Lewis and Gebhart, '77; Yaksh et al., '76). Simi- disrupts lordosis. Infusion of LHRH into the ventrolateral larly, responses to nociceptive neurons at the ventrobasal PAG failed to facilitate lordosis. Similarly, Sirinathsinghi thalamus to calibrated pinches are strongly depressed by ('84) has shown that microinfusion of gonadotropin-relasing micro-injection of morphine into the ventral PAG but not hormone, naloxone, or anti-(3-endorphin-globulin into the significantly changed when morphine is applied in the dor- dorsolateral PAG but not the ventrolateral PAG caused a sal PAG (Kayser et al., '83). Using a place-conditioning significant increase in lordosis behavior. Electrical stimu- paradigm Van der Kooy et al. ('82) have demonstrated pos- lation of the ventrolateral PAG, on the other hand, has itive reinforcement with morphine micro-injections into the been shown to suppress lordotic responsiveness in the fe- ventral PAG. These latter investigators also reported that male rat (Arendash and Gorski, '83). Taken together these injections of morphine into the ventral PAG produced pro- results suggest that the dorsolateral PAG may serve as a nounced analgesia in rodents as measured by the tail flick lordotic facilitatory region while the ventrolateral PAG may test, thus confirming previous reports. be involved in suppression of lordosis.

The fact that morphine exerts a greater effect in the The importance of the PAG in vocalization has been ventral PAG than in dorsal areas in the above behavioral known since the early work of Magoun and colleagues ('37). paradigms may be explained in part by recent data which Electrical stimulation of the PAG has been shown to elicit demonstrates that significantly higher number of p-opioid vocalization in the squirrel monkey (Jurgens and Pratt, binding sites are present in the ventral PAG as compared '791, rat (Waldbillig, '75), guinea pig (Martin, '761, and bat to the dorsal PAG (Moskowitz and Goodman, '84). Delta- (Suka et al., '73). The work of Jurgens and Pratt ('79) and opioid binding sites, on the other hand, were shown to be Larson and Kistler ('84) indicates that the dorsolateral PAG equally distributed between the dorsal and ventral PAG. plays a key role in the vocalization process. The latter The close correspondence among the distribution of p-opioid investigators, employing extracellular recording tech- binding sites, the site of initiation of analgesia resulting niques, have shown that the discharge of neurons in this from morphine micro-injection into the PAG, and our ven- region in all cases began prior to vocalization and in most trolateral subdivision provides both pharmacological and instances prior to any observable change in laryngeal EMG behavioral evidence to support the presence of this activity. These results suggest that the PAG may somehow subdivision. be involved in the initiation of vocalization.

Initial observations also suggested that the ventrolateral Finally, electrical stimulation applied to the dorsal part PAG was the most effective site for stimulation-produced of the PAG appears to have a strong aversive character and analgesia (SPA) (Dennis et al., '80; Lewis and Gebhart, '77; produces a vigorous escape response in the rat (Atrens et Mayer and Liebeskind, '74). Recent studies have docu- al., '77) and mouse (Cazala and Garrigues, '83) or leads to mented that SPA can be elicited from both the dorsal and aggressive behavior (Mos et al., '82a). The location of dorsal ventral regions of the PAG (Cannon et al., '82; Fardin et PAG sites which give rise to aversive reactions has been al., '84b; Gerhart et al., '84). However, Cannon and co- mapped by Fardin et al. ('84a). Micro-injections of the exci- workers ('82) have shown that systematically administered tatory amino acid glutamate into the midbrain PAG but naloxone consistently elevated SPA thresholds only in the not the hypothalamus of cats have been shown to induce a ventral PAG and was largely without effect dorsally. These rage reaction (Bandler, '82), while lesions of the PAG in data suggest that two pharmacologically separate analge- rats have been shown to decrease spontaneous aggression sia systems exist in this area, one located ventrally and one (Mos et al., '83). These studies support the role of midbrain dorsally. In support of this concept are data indicating that PAG neurons in defense-rage display and the role of the lesions of the raphe magnus disrupt SPA of the ventral dorsal PAG, in particular, in aversive reactions. PAG, as well as analgesia elicited by morphine micro-injec- Although it is not possible to equate every function that tion into this region, but not SPA from dorsal stimulation has been attributed to the PAG with a specific subdivision, sites (prieto et al., '83; Young et al., '84; see, however, there is considerable evidence to suggest that particular Gebhart et al., '83). Fardin and co-workers ('84a) have re- subdivisions play important roles in the many functions cently demonstrated that to obtain analgesia from dorsal discussed above. Thus the ventrolateral and dorsal divi-

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474 A.J. BEITZ AND R.D. SHEPARD

sions are involved in analgesia, while the dorsolateral sub- division appears to play a role in lordosis, vocalization, and aversive and aggressive behavior. The medial subdivision, which is the most distinct division anatomically, has re- cently alm been associated with aversive reactions (Fardin et al., '84a). Most investigations however, have ignored this small region. This may be due to the small size of the neurons that compose this region (Beitz, '85) as well as the small area occupied by this subdivision. Thus selective elec- trophysiological or chemical stimulation of this region is quite difficult. Future receptor binding, electrophysiologi- cal, and immunohistochemical studies may provide further evidence to support the presence of a medial subdivision.

ACKNOWLEDGMENTS The authors wish to thank Drs. David Brown and Thomas

Fletcher for their helpful suggestions during the prepara- tion of this paper. They also wish to thank Ms. Jane Spran- gers for typing the manuscript. This research was supported by grants BNS79-06486 and BNS83-11214 from the Na- tional Science Foundation.

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