A role of insular cortex in cardiovascular function

THE JOURNAL OF COMPARATIVE NEUROLOGY 257:189-207 (1987)

A Role of Insular Cortex in Cardiovascular Function

DAVID A. RUGGIERO, SIMA MRAOVITCH, ANTONIO R. GRANATA, MUHAMMAD ANWAR, AND DONALD J. REIS

Laboratory of Neurobiology, Cornell University Medical College, New York, New York 10021

ABSTRACT We sought to determine whether the insular cortex contributes to the

regulation of arterial blood pressure (AP). Responses to electrical and chemical stimulation of the cortex were studied in the anesthetized, paralyzed, and artificially ventilated Sprague-Dawley rat. The insular cortex was ini- tially defined, anatomically, by the distributions of retrogradely labeled perikarya following injections of wheat germ agglutinin-horseradish peroxidase (WGA-HRP) into the nucleus tractus solitarii (NTS). Injections of WGA- HRP into the insular cortex anterogradely labeled terminals in cardiopulmonary and other divisions of the NTS and confirmed projections revealed by retrograde tracing experiments.

Electrical stimulation of the insular cortex elicited elevations of AP (< 50 mm Hg) and cardioacceleration ( < 40 bpm). The locations of the most active pressor sites corresponded closely to the locations of retrogradely labeled cells in layer V of granular and posterior agranular areas of the insular cortex (areas 14 and 13) and the extreme capsule. Maximal pressor responses were obtained a t a stimulus intensity of three to five times threshold current of 20-30 PA. Responses elicited mostly with higher-threshold currents were also mapped in areas 2a and 51b and the claustrum and within the corpus callosum. Unilateral injections into the insular pressor area of the excitatory amino acid monosodium glutamate (L-Glu; 0.05 nmol to 10 nmol) or the rigid structural analogue of L-Glu, kainic acid (KA) (0.4 nmol) (which specifically excite perikarya), caused topographically specific elevations in AP and tachycardia.

During the course of the anatomical transport studies, new findings were obtained on the organization and characteristics of the cortical innervation of the NTS and the nucleus reticularis parvocellularis. Topographic relationships between the cortex and the NTS were organized in a more complex manner than previously thought. Cells projecting to caudal cardiopulmonary segments of the NTS were fewer and generally located ventrally and caudally and in a more restricted area than cells projecting rostrally or to the parvicellular reticular formation. Anterograde transport data revealed new presumptive terminal fields in dorsolateral, ventral, periventricular, and commissural regions of the NTS, including an area overlapping the terminal field of the aortic baroreceptor nerve.

We conclude that (1) neurons within an area of the insular cortex projecting to multiple brainstem autonomic nuclei, including a region of the NTS innervated by baroreceptor afferents, increase arterial blood pressure and heart rate; (2) cortical projections to the NTS are widespread but limited to discrete subdivisions of the nucleus; (3) a comparatively small percentage

Accepted September 3,1986.

0 1987 ALAN R. LISS, INC.

190 D.A. RUGGIERO ET AL.

of cortical neurons project to caudal versus rostra1 levels of the NTS; and (4) autonomic pathways from the insular cortex may regulate circulatory and possibly other autonomic functions.

Key words: nucleus tractus solitarii, arterial blood pressure, wheat germ agglutinin-horseradish peroxidase

For over a century the cerebral cortex has been known to play a role in autonomic regulation (see Hoff et al., '63). More recently, electrical stimulation of a broad area of the cortex, encompassing the orbitofrontal area, cingular region, insular-temporal lobe, and sensorimotor cortex, has elicited alterations in autonomic activity in several species (Crouch and Thompson, '39; Hoff and Green, '36; Green and Hoff, '37; Kennard, '45; Kaada, '51; Wall and Davis, '51; Eliasson et al., '52; Delgado, '60; Hoff et al., '63; Clarke et al., '68). These alterations included changes in respiration and heart rate (HR), blood flow, gastrointestinal motility, pilo- and sudomotor responsivity, and both increases and falls in arterial blood pressure (AP). Attempts to interpret these data, however, have been problematic, mainly because of inconsistent and contradictory results, the diffi- culty of comparing observations reported in different species, and, with few exceptions (Wall and Davis, '51; Eliasson et al., '521, the lack of direct correlations between physiological responses and anatomical pathways. The techniques previously employed to stimulate the brain, moreover, did not allow more precise topographic localization of the critical area of cardiovascular responsivity. At present, large gaps remain in our understanding of the precise cortical areas and pathways mediating autonomic function. The autonomic functions of the cerebral cortex, for example, have not recently been systematically mapped with contemporary techniques and no data are available on our experimental model, the rat.

Neurons in the insular area, one of the regions from which autonomic responses are elicited (see references above), have recently been shown to innervate nuclei that play a role in autonomic control including, several sub- structures in the subcortical forebrain, parabrachial complex, medullary reticular formation, and the nucleus tractus solitarii (NTS) (Ross et al., '81; Mraovitch et al., '82; Saper, '82a; Shipley, '82; van der Kooy et al., '82). These anatomical connections involving structures linked to cardiovascular regulation therefore raise the possibility that one of the functions of the insular cortex might be a role in the regulation of AP. In the present st.udy we sought to determine in rats whether electrical stimulation of the insular cortex elicits cardiovascular responses, and if so, whether there is a correlation between sites of autonomic projection and those of cardiovascular responsivity. During the course of this study, we also sought to reevaluate the organization and characteristics of the cortical innervation of the NTS.

MATERIALS AND METHODS Anatomical studies

Studies were performed with 45 male Sprague-Dawley rats anesthetized with halothane (2% in 100% 0 2 ) and mounted in a stereotaxic apparatus (Kopf) with the bite bar set at - 5 . In most animals injections of 1-5% solutions of wheat germ agglutinin-horseradish peroxidase conjugate (WGA-HRP) were made stereotaxically with the aid of an

injection carrier upon which a 1-p1 Hamilton syringe was mounted or via a calibrated micropipette (50-pm tip diameter) attached (via cannula tubing) to an air-pressure injection system.

After 24-72 hours, animals were reanesthetized and perfused transcardially with saline, 0.4% paraformaldehyde- 1.25% glutaraldehyde in phosphate buffer followed by a 15% sucrose solution. The entire brain and representative segments of spinal cord were sectioned coronally at 35 pm. Sections at approximately 105-pm intervals were processed by using tetramethylbenzidine (TMB) to demonstrate the injection sites and the presence of labeled processes and perikarya. Alternate sections through the injection sites were also treated with diaminobenzidine (DAB) and com- pared to adjacent TMB-processed tissues in order to approximate the central core of the injection deposit surrounding the micropipette tip, and counterstained with cresyl violet or thionine to establish nuclear boundaries (see Ruggiero et al., '82). Computer reconstructions of the TMB-processed injection sites consistently reveal a dense central core and a diffusion gradient of gradually decreasing reaction density (see Milner et al., '86). In our material, effective transport appeared to be limited to the dense central core and immediate surround but not the outer radius of diffusion. The size of the injection was found to be dependent on the volume and concentration of the tracer, survival time (the diffusion sphere decreases following longer survival pe- riods), length of the dehydration procedure, and sensitivity of the TMB reaction.

In the first series of experiments, the patterns of retrograde transport to the cerebral cortex were analyzed in 20 cases in which WGA-HRP was microinjected into representative rostrocaudal levels of the NTS or adjacent aspects of underlying reticular formation. Most injection sites were unilateral, although injections made into the caudal commissural NTS were usually bilateral. In general, volumes of 3-5 nl of 5% WGA-HRP resulted in relatively small injection sites, restricted to the NTS, and were especially useful for analyzing topographic relationships. Larger injections (10-20 nl) of less concentrated WGA-HRP (1%) were also analyzed. Camera lucida drawings of representative injection sites appear in Figure 1; photomicrographs appear in Figure 2. Control injections of similar volumes of WGA- HRP were also placed into the ventrolateral medulla, dorsal column nuclei, hypoglossal nucleus, and the vestibular complex. Data were also available from animals in which multiple, more concentrated volumes of WGA-HRP (5-10%) were injected into thoracic or lumbosacral spinal segments.

In a second series of experiments (n=25), unilateral (A226, A252, A342, A371, A690) or bilateral (A679, A692, A733, A742) injections of 1-5% WGA-HRP were slowly made into areas 13 and/or 14 of the cerebral cortex over a period of 15-30 minutes, after which the needle or pipette was left in place for another 10-30 minutes. In these animals the distributions of anterogradely labeled processes in the NTS (and in other areas of medulla) were mapped. In order to

INSULAR CORTEX AND AUTONOMIC FUNCTION

maximize anterograde labeling of terminals in the NTS, in several animals (A694, A764, A7651 multiple injections of 5-15 nl of WGA-HRP were placed bilaterally along the rostrocaudal axis of the insular cortex. Although several of the larger injection sites involved areas bordering the insular region (for example, primary olfactory cortex or the frontoparietal somatosensory area), these regions, based on our control data and those of others (Saper, '82; Shipley, '82; van der Kooy, '821, do not project to the NTS. Control injections were placed in the following regions adjacent to the insular cortex: prepyriform cortex, somatosensory areas (I and/or 111, perirhinal cortex (caudal to the retrogradely labeled field), anterior portions of the ventral and dorsal agranular insular areas, claustrum, or caudoputamen. Other injection sites involving the cingulate and precentral cortex were also available. After injections into the insular cortex, segments of cervical, thoracic, and lumbosacral spinal cord were also examined for anterograde transport. In all experiments section outlines were drawn by using a Bausch and Lomb overhead projector; labeled cells and processes and nuclear borders were mapped with a camera lucida attached to a light microscope. Because the entire brainstem is often processed in our studies, material from cases in this investigation has already been described or will serve as a source of data for future studies.

Physiological studies Preparation of animals. Experiments were performed

on a total of 15 Sprague-Dawley rats (300-350 g) anesthetized with 2% halothane. Plastic (Tygon) catheters were inserted into the left femoral artery for recording arterial pressure (AP) and into the left femoral vein for administra- tion of drugs. The trachea was cannulated and the animal was placed in a stereotaxic frame. The parietal bone was perforated ipsilaterally by a dental drill at points 5 mm anterior to the interaural line and 4 mm lateral to the midline. The drill holes were then enlarged laterally to approximately 6 mm from the midline and anteriorly to 11.0 mm from the interaural line. The dura were then reflected and the surface of the brain was kept warm and moist throughout the experiment. An intravenous injection of alpha-chloralose (40 mgkg) was made; the halothane was discontinued; and the animal was then paralyzed with gal- lamine triethiodide (Flaxedil, Davis and Geck; 5 mgkg i.v.1 and artificially ventilated by a respirator pump. Body tem- perature was maintained at 37°C by a heating pad.

Electrical and chemical stimulation of the insular cortex. The insular cortex and adjacent cortical areas were systematically explored for a cardiovascular response to electrical stimulation through a monopolar electrode. Elec- trodes were fabricated from 70% platinum, 30% iridium wire (150 pm in diameter) and insulated by Epoxylite ex- cept for the bare tip. Electrodes were inserted vertically into the brain. The area that was explored for cardiovascular responses extended from the interaural line, with reference to coordinates of the rat's brain as defined by Pellegrino et al. ('67), rostrocaudally from A10.5 to A 4.5 and laterally from L4.0 to L6.3. Vertically, the tracks were explored, in steps of 0.5 mm, between the surface of the cortex (H=O.O) and H -7.5 mm.

The brain was stimulated with an 8-second train of nega- tive square-wave pulses of 0.5-msec duration at a stimulus frequency of 50 Hz delivered to the animal from a pulse generator (Ortec model 4656). The stimulus current, mea- sured by passing the stimulus across a 104 resistor, was

191

amplified by a Grass P15 AC preamplifier, displayed on a cathode ray oscilloscope, and monitored throughout the experiment. Stimulation was monopolar via an alligator clip anode attached to exposed neck muscle. For identification of stimulating sites, each tract was marked with a small lesion a t two spots by a 15-second direct current of 50 pA.

Unilateral microinjections of excitatory amino acids, L- glutamic acid (L-Glu) or kainic acid (KA) (which excite neuronal perikarya but not axons), were performed in six

A bbreuiations

AC ACC AHA AP BL cc CE CL CNV CP D

DAO DL ECN F FR GP HPC I IC IVN LRN LSN M MA0 MD MLF MPO MSN M l T MVN NC NG NGCd NGCv NPS

NPvc NTSc NTSi NTSr NST PION PP RD RF RM RV RVL

STNc STNi STNo STT TO TUO Vm

VB VL VM ZI X XI1

anterior commissure nucleus accumbens anterior hypothalamic area area postrema basolateral nucleus of amygdala corpus callosum (or central canal) central nucleus of amygdala claustrum commissural nucleus of vams (also NTSc) - caudate-putamen dorsal division (terminal field) of commissural NTS (contiguous with DL) dorsal accessory olive dorsolateral division (terminal field) of NTS external cuneate nucleus fornix rhinal fissure globus pallidus hippocampal commissure intermediate division of NTS nucleus intercalatus inferior vestibular nucleus lateral reticular nucleus lateral septal nucleus medial division of NTS medial accessory olive mediodorsal thalamic nucleus medial longitudinal fasciculus medial preoptic nucleus medial septal nucleus mammillothalamic tract medial vestibular nucleus nucleus cuneatus nucleus gracilis nucleus reticularis gigantocellularis pars dorsalis nucleus reticularis gigantocellularis pars ventralis nucleus parasolitarius (lateral to DL or dorsal to D and defined by the terminal field of the fastigial cerebellar nucleus nucleus reticularis parvocellularis nucleus tractus solitarii (caudal third) nucleus tractus solitarii (intermediate third) nucleus tractus solitarii (rostra1 third) nucleus of the stria terminalis principal inferior olivary nucleus nucleus prepositus nucleus reticularis dorsalis retrofacial nucleus nucleus raphe magnus nucleus reticularis ventralis nucleus reticularis rostroventrolateralis (defined by Ross et al., '84a, '85; Ruggiero et al. '85) spinal trigeminal nucleus pars caudalis spinal trigeminal nucleus pars intermedius spinal trigeminal nucleus pars oralis spinal trigeminal tract olfactory tract olfactory tubercle ventromedial "arcuate" division (terminal field) of NTS (contiguous with ventromedial division of CNV) ventrobasal thalamic nucleus ventrolateral division of NTS ventromedial thalamic nucleus zona incerta dorsal motor nucleus of vagus hypoglossal nucleus


D

Figure 1A-D

E A13

Fig. 1. Camera lucida drawings of transverse sections of medulla demonstrating WGA-HRP injection sites in the nucleus tractus solitarii (NTS) and underlying reticular formation. Stippling gradients illustrate relative densities of TMB-processed injection sites. A-C: Injections centered in the rostral one-third of NTS. Portions of the medial vestibular nucleus were involved in A78 and to a lesser extent in A689 but not in A751, although

the pattern of transport to insular cortex was consistent throughout. D: An injection centered in the nucleus reticularis parvocellularis. E-H: Injections centered on caudal and intermediate thirds of NTS. Control injections were also made in the dorsal column nuclei, hypoglossal nucleus, vestibular complex, nucleus reticularis gigantocellularis, the rostral ventrolateral medulla, and the thoracic and lumbosacral spinal cord (not illustrated).


Fig. 2. Bright- and darkfield photomicrographs of injection sites restricted to the nucleus tractus solitarii (NTS). A,C: Examples of two injection sites processed with diaminobenzidine (DAB) centered on the rostra1 (A7511 and intermediate (A7441 thirds of the NTS and counterstained with cresyl violet. B,D: Higher power views ofthe same injection sites processed with TMB on sections taken adjacent to A and C, respectively. E,F: Injec- tion sites processed with TMB, centered on the subpostremal (A688) and

commissural (A694) regions of the caudal NTS. Data combined from tissues processed by both TMB and DAB were used to evaluate sites from which effective transport occurred. Effective transport was limited to the dense core and immediate surround but not the periphery of the TMB-processed injection site. Computer reconstruction of the TMB field reveals a dense inner core and a gradient of gradually decreasing reaction density (see Milner et al., '86). B a r = l mm (A$) or 380 pm (B,D,E,F).

experiments. Effective injections were made into an area of insular cortex from which maximal cardiovascular responses were elicited by electrical stimulation. Control microinjections were made l mm dorsal or l mm ventral to this area. L-Glu or KA was dissolved in phosphate-buffered saline (pH 7.4) and injected into the cortex by using glass micropipettes (approximately 50 pm in outer diameter). The position of the micropipette in the insular cortex was determined postmortem by localization of fast green dye co-

injected with the excitatory amino acids. Histological examination. At the end of each experi-

ment, the brain was perfused through the heart with 0.9% NaCl solution followed by 10% formaldehyde solution; fro- zen sections of 40 pm were then cut on a sledge microtome and stained with cresyl violet for histological examination. The stimulus and microinjection sites were reconstructed in relation to histological structures with the aid of an overhead projector.

INSULAR CORTEX AND AUTONOMIC FUNCTION 195

RESULTS Anatomical localization of autonomic cortex

Anatomical experiments were used to first localize sites in cerebral cortex defined on the basis of interconnections with the nucleus tractus solitarii (NTS).

Retmgrade transport studies. In the first series of experiments, WGA-HRP was used as a retrograde tracer to locate sites in cortex containing the highest concentration of neurons projecting to the NTS. Microinjections of WGA-HRP were placed in individual rostral-caudal subdivisions of NTS (Figs. 1, 2) and the distributions of retrogradely labeled cells were mapped in cerebral cortex. Data are presented from cases A78, A63, and A13, which represent small injection sites that fairly selectively involved rostral, intermediate, or caudal parts of the NTS (Figs. 1,3). The latter two injections were centered on an area of the NTS that is heavily innervated by primary baroreceptor and cardiopulmonary afferents. In each case, there was minimal spread of the tracer to the adjacent rostral or caudal subdivisions of the NTS and dorsal motor nucleus. The nucleus gracilis was involved in case A13 (and minimally in A63) and the WGA-HRP leaked into the vestibular complex in case A78. Similar findings were obtained from injection sites involving primarily caudal andor intermediate levels of the NTS (A680, A684, A688, A693, A694, A744) or the rostral one- third of the nucleus (A686, A689, A751) (Figs. 1, 2, 4). In separate experiments, control injections were made into areas adjacent to the NTS including the nucleus reticularis parvocellularis (NPvc) (Figs. 1,3), nucleus gigantocellularis pars dorsalis, dorsal column nuclei, medial and inferior vestibular nuclei, and hypoglossal nucleus (not illustrated). Data were also available from animals in which WGA-HRP was injected into the rostral ventrolateral medulla or segments of the thoracic or lumbosacral spinal cord.

After injections of WGA-HRP into the NTS (or NPvc), transport occurred bilaterally with contralateral predominance to cells of layer V primarily in area 14 and within the dorsal aspect of area 13-regions respectively termed the dorsal and ventral insular cortex by Krieg ('46). Cytoar- chitecturally these regions are equivalent, respectively, to the more recently defined granular and posterior agranular areas of Krettek and Price ('77), subdivisions of the claus- trocortex of Zilles et al. ('801, or the gustatory and caudal prefrontal cortical regions of Gulden and Markowitsch ('83). Unless indicated otherwise, the terminology of Krieg ('46) and Krettek and Price ('77) will be used for reference.

Following small injections centered in the rostral one- third of the NTS (the gustatory field), the largest number of labeled cells occurred in the contralateral insular cortex. The majority of labeled cells extended from the level of and rostral to the decussation of the anterior commissure (Fig.

Injections into caudal levels of the NTS, where afferents from cardiopulmonary receptors preferentially terminate, resulted in a considerably smaller percentage of labeled cells in the insular cortex (Fig. 3A,B). After caudal injections, cells in insular cortex were also labeled primarily contralateral to the site of injection, although their distribution was more limited (restricted to ventral aspects of layer V) and not as extensive rostrocaudally. Most neurons were labeled anterior to but not at the level of or caudal to the decussation of the anterior commissure. Similar topographic data were obtained after larger injections into ros-

3A-C).

tral or caudal levels of the NTS, although in these cases a larger number of more intensely labeled cells were seen. Photomicrographs comparing the distribution of labeled cells in the insular cortex were taken from cases A689 and A680 and are provided in Figure 4.

Control injections into the dorsal aspect of NPvc (Fig. l), variably involving the nucleus cuneatus andor the spinal trigeminal nucleus, resulted in a pattern of transport comparable to that seen after injections into the rostral NTS (Fig. 3A-C). While some overlap occurred within the ventral part of area 14, cells projecting to the reticular formation were more widely dispersed and extensive involving the dorsal aspect of area 14 and area 2a (somatosensory area I1 of Krettek and Price, '77; sensory areas Sm2 and adjoining ventral portions of Sml of Zilles et al., '80). Con- trol injections centered in the nucleus gracilis transported to sensory area 3 (somatosensory area I). Labeling of area 3 also followed injections into the commissural NTS, and, in keeping with control data, was proportional to the degree to which the tracer diffused into the nucleus gracilis. Injec- tions of WGA-HRP into a sympathoexcitatory area of the rostral ventrolateral medulla (Ross et al., '84a,b), the medial and inferior vestibular nuclei overlying the rostral NTS, the hypoglossal nucleus, or the thoracic or lumbosacral spinal cord did not result in transport to the insular cortex.

Antemgrade transport studies. In order to confirm and extend retrograde transport data, we next sought to map the distribution of afYerents to the NTS by injecting WGA- HRP into granular and agranular fields of the posterior insular cortex (areas 14 and 13 of Krieg, '46). In these experiments WGA-HRP was used as an anterograde tracer. Most injections were placed unilaterally and employed 1% solutions of WGA-HRP. Bilateral injections, or injections employing higher concentrations of the tracer, were also made in order to enhance transport and improve visualiza- tion of the labeled fields (particularly in the caudal NTS where terminals tended to be less concentrated and lightly labeled). The following description is based on two representative cases (A226 and A694; Figs. 5, 6). In A226, a large unilateral injection of WGA-HRP was centered in areas 13 and 14 with some spread of the tracer into the surrounding somatosensory cortex and claustrum. A commissural projection, via the ventral corpus callosum, mirrored the dense central core of the injection site and terminated within the homologous locus in the contralateral cortex. Perikarya were also labeled in equivalent commissural and associa- tional areas of the insular region (including the perirhinal and lateral prefrontal fields) but are not illustrated. In A694, where multiple bilateral injections of WGA-HRP involved the insular cortex and to a lesser degree the adjacent somatosensory area, transport was considerably heavier. Smaller, more restricted injections into the insular cortex resulted in an identical pattern of transport to the NTS.

Labeled processes extended from the rostral pole of the NTS to the commissural nucleus of the vagus (Fig. 5A-E). In the rostral NTS (Fig. 5A), presumptive terminals were labeled throughout the division with particularly dense projections to ventrolateral, dorsal, and dorsomedial locations.

At an adjacent level, where the NTS approaches and contributes to the floor of the fourth ventricle (Fig. 5B), labeled terminals surrounded the solitary tract and were

196 A ROSTRAL D.A. RUGGIERO ET AL.

0 NTSi (A63) . NTSr iA78)

Fig. 3. Camera lucida drawings demonstrating the anatomic Iocalization of an area of autonomic representation in insular cortex based on the distributions of retrogradely labeled perikarya following injections of wheat germ agglutinin-horseradish peroxidase conjugate (WGA-HRP) into the NTS and the NPvc. The distributions of labeled cells are bilateral yet predominantly contralateral and localized primarily to areas 14 and 13 of Kreig (’46). Larger numbers of cells were laheled after injections into the rostral

NTS or NPvc and concentrated in a region of frontal cortex lying at the level of and just rostral to the decussation of the anterior commissure. Cells labeled from the caudal NTS were more restricted to ventral and caudal (but not caudalmost) aspects of insular cortex. After NTS injections, another smaller group of cells was labeled in prelimbic and infralimbic areas of cortex as described by van der Kooy et al. (‘82) and Terreberry and Neafsey (‘83), although these were not investigated in this study.


ROSTRAL NTS A 689

197

CAUDAL NTS A 680

Fig. 4. Camera lucida drawings and corresponding darkfield photomicrographs showing retrograde transport of WGA-HRP to insular cortex after injections into NTS. The injection into the rostral third of NTS also spread into the overlying medial vestibular nucleus (A689); injections into the

caudal NTS were bilateral and centered at and just caudal to the level of the obex (A680). Note the more extensive distribution of cells labeled after the rostral injection vs. the more restricted ventral localization after caudal injections. Bar = 50 pm.

198 A226 D.A. RUGGIERO ET MA.

Fig. 5. Camera lucida drawings of a large injection site centered in frontal insular (and adjacent somatosensory) cortex, and transport of the tracer, WGA-HRP, to terminals and fibers in passage in the NTS. Most terminals are concentrated in rostra1 aspects of the NTS and progressively decrease in density a t caudal levels (A,B). Terminals were also densely labeled in the ventrolateral pulmonary subdivision (VL) and to a lesser extent in a baroreceptor afferent terminal field (see Ciriello, '83) in close proximity to the tract, including dorsolateral, intermediate, and commis-

sural subdivisions (C-E). Terminals in the dorsolateral division on one side of the subpostremal aspect of NTS merge with the contralateral field and form a strip lining the dorsal border of the commissural nucleus (El. Note also the presence of terminals in adjacent aspects of NPvc and the spinal trigeminal complex. Control injections into adjacent areas including the somatosensory, perirhinal, or piriform cortex, the claustrum, and the putamen did not label the NTS and verify the specificity of these projections from the insular cortex.


D

E

Figure 5 continued

concentrated ventrolaterally (within ventrolateral, intersti- tial, intermediate, and ventral divisions) and dorsomedially (within an area we term the medial parvicellular field and which appears to correspond to the dorsal aspect of the medial subnucleus of Kalia and Sullivan ('82). A dense strip of terminals also occurred, at this level, in the periventricular gray subadjacent to the floor of the fourth ventricle; some of these fibers extended medially and decussated across the midline. On some sections, these terminals were contiguous, laterally, with another labeled strip outlining the dorsal and dorsolateral border of the NTS. In ventral and ventromedial areas of NTS (medial to and contiguous with the ventral subnucleus of Kalia and co-workers, '84,

'85), processes were found surrounding and lying within the body of the dorsal motor nucleus.

At the level of and just rostra1 to the area postrema (Fig. 5C,D), a contiguous field of light to moderately labeled terminals was present in a region of the NTS innervated by primary cardiopulmonary derents; these terminals were distributed adjacent to the tract within dorsolateral (including the dorsal, dorsolateral, and dorsal strip regons of Kalia et al., '84, '85i, intermediate, ventral, and ventrolateral regions. Terminals in ventral and ventromedial aspects of the NTS formed an arc surrounding the dorsal motor nucleus and extended ventrally below it and dorsal to the nucleus intercalatus; other labeled fibers were iden-

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Fig. 7. Changes in arterial pressure (AP) and heart rate (HR) elicited by electrical stimulation of the insular cortex of an anesthetized rat. Stimulus frequency and stimulus intensity response characteristics. A Drawing of a coronal section of the frontal forebrain at the level of the anterior commissure’s decussation (A 7 k 0.5). An electrode track that penetrates the cortex is shown with stimulus sites (a-o) illustrated by filled circles 500 Prn apart. At each site the cortex was stimulated with an 8-second stimulus train of

square-wave pulses (0.5-msec pulse duration, 50 HZ at 100 PA). B: AP, mean arterial pressure (MAP), and HR responses of the stimulus sites. C Effects of stimulus frequency upon pressor responses elicited in the insular cortex expressed as a change in AP (mmHg) from the prestimulus level. The stimulus parameters are the same as in A and B (n=4). D: Effect of stimulus intensity upon pressor responses elicited in the insular cortex. The stimulus parameters are the same as in A and B (n=4). Values are mean f SEM.

titled in close proximity to the central canal. At this level, few or no terminals were labeled centrally or further medially within the medial or commissural divisions (as defined by Kalia and Sullivan, ’82) of the complex, and none were seen in the area of postrema.

At the commissural level of NTS (caudal to the area postrema) (Fig. 5E), terminals decreased in density but were contiguous with those labeled further rostrally. The dorsolateral terminal fields of both nuclei rotated medially, fused, and formed a horizontal strip of relatively lightly labeled terminals within the dorsal NTS; these were contiguous across the midline and apposed dorsally to the dorsal column nuclei. The ventral field of terminals surrounding the dorsal motor nucleus, extended, laterally, toward the tractus solitarius and ventrolateral to it and, medially, adjacent to the central canal. Photomicrographs of labeled processes in NTS are provided in Figure 6. In addition to labeling of the NTS, heavy transport occurred to terminals in the nucleus reticularis parvocellularis (or at caudal levels, the nucleus reticularis dorsalis) and to the spinal trigeminal nucleus, with especially dense innervation of its peripheral division, pars zonale. Terminals in the reticular formation were concentrated dorsally in direct contiguity with those labeled in the ventrolateral NTS and extended ventrally as far as the nucleus ambiguus. No anterograde transport was found in the intermediolateral or intermedi-

omedial cell columns of the thoracic spinal cord or (on the basis of data from cases A764 and A765) at lumbar or sacral spinal levels.

Effect of electrical or chemical stimulation of insular cortex on arterial pressure and heart rate Electrical stimulation. In anesthetized, paralyzed rats,

electrical stimulation localized within the anatomically defined region of the insular cortex (areas 13 and 14) elicited a rise in arterial blood pressure (AP) and heart rate (HR). For convenience, the response has been termed the insular pressor response (IPR) and a representative experiment is illustrated in Figure 7A and B. An electrode was lowered through the frontal cortex in 0.5-mm steps, and at each point an 8-second stimulus train of square-wave pulses (0.5- msec duration, 50 Hz and 100 PA) was delivered. Hyperten- sive responses appeared when the electrode entered the insular cortex (Fig. 7 A,B; i-m). The rise in AP (< 50 mmHg) occurred within 2-3 seconds of the onset of the stimulus train, was sustained during the stimulation, and rapidly recovered to control levels after the stimulation was terminated. Cardioacceleration (10-20 bpm) was coupled to the rise in AP.

General characteristics of pressor responses from the insular cortex. The relationship between frequency and intensity of the stimulus and magnitude of the pressor

D.A. RUGGIERO ET AL. 202

T U O

1 m m Threshold cu r ren t <50@ Threshold curren t >50-100@

I No pressor response

response was studied in four rats. When the stimulus frequency was varied at a stimulus intensity of 100 @A (Fig. 7C), the response appeared at 5 Hz and reached maximum between 50 Hz and 100 Hz. When the current intensity was varied, keeping the stimulus frequency at 50 Hz (Fig. 7D), the elevation of AP (equal to 10 mmHg) (threshold current) appeared with a stimulus current ranging between 20 and 30 PA. Blood pressure increased in proportion to the magnitude of the stimulus intensity (Fig. 7D); the maximum pressor response was obtained at a stimulus intensity of three to five times the threshold.

Localization of pressor sites in the insular area. A systematic exploration of the insular area and adjacent areas

Fig. 8. Systematic determination of the sites a t which pressor responses of 10 mmHg were elicited by the lowest stimulus current-the threshold current-in insular cortex and adjacent areas in rat brain. The cortex was stimulated every 500 pm from the surface with an 8-second train of square- wave pulses (0.5-msec pulse duration, 50 Hz) and the threshold current was determined at each site. Lowest thresholds (4 50 PA) for responses are represented by large filled circles and higher thresholds ( > 50-100 pA) by medium-sized filled circles. Small dots represent sites yielding no pressor response. Explored sites were grouped on six coronal sections. The data illustrated are a composite of representative tracts obtained from five rats. Note that active sites yielding maximal pressor responses are localized, with few exceptions, within the anatomical boundary of the insular cortex.

of cerebral cortex in the rat was undertaken to localize sites from which increases in AP and HR were elicited by electrical stimulation with optimal parameters. Positive sites were defined as a point along an electrode tract from which a barely detectable change in AP was elicited with threshold current.

Systematic exploration of the positive sites at which pressor responses were elicited involved over 50 tracks (Fig. 8).


Fig. 9. Effect of unilateral microinjection of L-glutamic acid CL-Glu) into the left insular cortex upon the arterial pressure and heart rate of an anesthetized rat. A: Drawing of a coronal section of the frontal forebrain rostra1 to the anterior commissure's decussation (A9 0.5), illustrating the

Stimulation consisted of an 8-second train of rectangular pulses of 0.5 msec delivered at 70 Hz (optimal frequency for the insular cortex stimulation); the threshold current was determined at each site. Explored sites were graphed on six coronal sections from A 10 _+ 5 to A 5 f 5 from the interaural line (Fig. 8A-F). The distribution of sites at which the threshold current for the pressor response was less than 50 pA was largely concentrated within areas 13 and 14 of Kreig, and extended from levels A9 & 0.5 to A 6 k 0.5 (Fig. 8B-El. Most positive sites were encountered in deep aspects of the cortex and within the extreme capsule adjacent to the claustrum. A few positive sites (Fig. 8C,D), most having high thresholds, were found in areas 2a and 51b and the claustrum and within the corpus callosum.

Chemical stimulation. In another experimental group of four rats anesthetized with chloralose (100 mgkg i.v.1, a unilateral microinjection into the insular area of L-glutamic acid (L-Glu; 0.05-10 nmol dissolved in 100 nl of saline) elicited a moderate increase in AP (19 4 m a g ) and HR (40 f 5 bpm) (values are mean * SEW. The results of a typical experiment illustrated in Figure 9 demonstrate that after the injection of L-Glu, AP increases with maximum values observed approximately 15 minutes after the injection.

In two animals, KA (400 pmol dissolved in 100 nl of saline) microinjected into the insular area elicited similar increases in AP and HR. Unilateral microinjections of ve- hicle did not change resting levels of AP or HR. Fast green dye (1%) was co-injected with L-Glu in order to determine the area involved by the injection.

Fast green dye (1%) was restricted to the insular cortex just lateral to the claustrum at the level of the anterior commissure decussation in an area from which electrical stimulation elicited maximum changes in AP. Microinjec- tions of L-Glu into more dorsal or ventral areas did not change resting AP or HR.

approximate site of a typical injection (marked by an injection of 100 nl of dye (stippled area) into the left insular cortex. B a: Cardiovascular effect of a unilateral microinjection of L-Glu (10 nmol in 100 nl of saline) into the left insular cortex. b: Maximum effect obtained after 15 minutes.

DISCUSSION The present study demonstrates that electrical or chemi-

cal stimulation of a restricted region of insular cortex in rat elicits a moderate elevation of arterial blood pressure and heart rate that we have termed the insular pressor response (IPR). The specificity of the IPR is supported, first, by the observation that pressor points having the lowest threshold were consistently localized to a discrete cortical area and were in close proximity to unresponsive sites (e.g., striatum) or areas responding only to high-threshold stimuli (e.g., somatosensory cortex). In addition, meningeal stimulation (a potential source of error) elicited depressor responses but never a rise in arterial pressure.

The pressor response produced by electrical stimulation of the insular region, moreover, appears to occur through excitation of intrinsic perikarya. Thus, local microinjections of the excitatory amino acids L-Glu or the rigid structural analogue of L-Glu, kainate, elicited an IPR similar in location and comparable in magnitude to that produced by electrical stimulation. Since these amino acids when applied in brain excite perikarya and not s o n s (Zieglgansber- ger and Puil, '73; Fries and Zieglgansberger, '74; Nicoll et al., '80; Millar and Armstrong-James, '821, the IPR can be attributed, at least in part, to excitation of cells intrinsic to the insular region. Experiments with excitatory amino acids, of course, do not rule out the possibility that some of the pressor activity localized electrically might also have resulted from stimulating axons. This point is supported by the observation that low-threshold pressor sites also occurred posterior or medial (deep) to the locations of labeled cell bodies in areas through which axons from the insular cortex pass. Intracortical fibers from autonomic projection cells in deep layers of the posterior insular region, for example, project medially toward the internal capsule and subcortical forebrain and rostrocaudally through the ventrolateral prefrontal and perirhinal (suprarhinal) areas


(Saper, '82a). An area where pressor sites are densely concentrated, between layer VI and the claustrum (the extreme capsule), is also traversed by fibers originating from other cortical areas including the anterior agranular cortex (Reep and Winans, '82) and by afl'erents derived from subcortical nuclei (e.g., the dorsomedial nucleus of the thalamus) (Krettek and Price, '77).

The region of cortex from which maximal cardiovascular responses were elicited is identified anatomically by its widespread projections to brainstem nuclei having critical and widely recognized roles in autonomic function including those related to cardiovascular regulation (Ross et al., '81; Mraovitch et al., '82a,b; Saper, '82a,b; van der Kooy et al,, '82). By cross-comparing our anatomical transport and physiological data we have recognized a close approxima- tion between the insular pressor area and the origin of the cortical innervation of two of these projection nuclei: the NTS (the medullary relay for primary visceral afferents) and the NPvc (a sympathoexcitatory area of medullary reticular formation [Dampney et al., '79; Goodchild and Dampney, '851 and a component of the more generalized propriobulbar field [Holstege and Kuypers, '77; Holstege et al., '77; Ruggiero et al., '821).

The pathways mediating the IPR are a t this point un- known, although recent anatomical evidence indicates that insular corticobulbar fibers course by two principle routes- one via the pyramidal tract and another via the medial forebrain bundle that is contiguous, in the lower brainstem, with the central tegmental field (Saper, '82a; van der Kooy et al., '82). Both pathways were confirmed in our material. Of the structures innervated by the insular pressor area, many have long been associated with circulatory and other visceral reflex functions, and therefore represent potential relays for the IPR. These sites include nuclei of the subcortical forebrain, such as the amygdala or hypothalamus, the parabrachial complex, the NPvc, and the NTS (e.g., Alex- ander, '46; Doba and Reis, '73; Dampney et al., '79; Hamil- ton et al., '81; Ross et al., '81; Mraovitch et al., '82a,b; Saper, '82a,b; Shipley, '82; van der Kooy et al., '82). A direct projection to autonomic spinal neurons, as suggested by Shipley ('82) and more recently by Shimada et al. ('851, may mediate the IPR, although we have not been able to confirm this pathway with either retrograde or anterograde axonal tracers. Injections of WGA-HRP placed throughout the thoracic or lumbosacral spinal cord, for example, consistently failed to retrogradely label cells in the insular pressor area; moreover, no fibers could be traced into the white matter of the cervical spinal cord or the intermediolateral or inter- mediomedial cell columns after injections of the same tracer into the insular cortex (this study). In our recent experiments (A764, A765) bilateral injections of WGA-HRP centered on the insular cortex and minimally involving the somatosensory areas labeled terminals in the dorsal horn and lamina X of upper cervical cord. These, however, were in contiguity with the terminal fields traced from the dorsal medulla. Based on the work of Shimada et al. ('85) neurons in the insular cortex (in an area primarily caudal to the NTS projection cells) were retrogradely labeled after injections of biotin-HRP into lumbar and upper cervical levels of the spinal cord. Whereas their photomicrographs (see their Fig. 2) suggest that these cortical neurons are sparse and weakly labeled, more extensive studies will be needed to define their precise terminal fields as well as their func- tional significance.

The insular cortex does not directly project into the RVL, an area innervating autonomic spinal neurons and playing a critical role in the tonic and reflex modulation of arterial blood pressure (Amendt et al., '79; Dampney et al., '82; Granata et al., '83, '85; Ross et al., '84a,b, '85). If autonomic neurons in the RVL and spinal cord do play a part in the IPR, the mechanism could occur indirectly via relays to these structures from regions innervated by the insular cortex such as the hypothalamus, parabrachial complex and NTS (Saper et. al., '76; Loewy and Burton, '78; Saper and Loewy, '80; Andrezik et al., '81; Ruggiero et al., '84).

During the course of this study new anatomical findings were obtained on the organization and characteristics of the cortical innervation of the NTS. Prior to this report, the corticosolitary projection was demonstrated, on the basis of anterograde transport data, to be bilateral and ordered viscerotopically in a rostrocaudal sequence (Saper, '82a; Shipley, '82). Our studies, based on both retrograde and anterograde transport methods, confirm and extend these findings. Cortical afferents to the NTS were distributed bilaterally with contralateral predominance as described by Saper ('82), although in contrast to the mainly ipsilateral projections reported in mouse (Shipley, '82) and rat (van der Kooy et al., '82). Based on our retrograde transport data, we found that cells projecting to caudal cardiopulmonary segments of the NTS were comparatively small in number and generally located ventrally and (as described in the above reports) caudally in insular layer V. Neurons projecting to rostral "taste" regions of NTS, on the other hand, overlapped cells projecting to caudal levels, but were also more extensively derived from dorsal and rostral as well as caudal cortical sites. The latter observation was consistently supported by our retrograde transport data and by the fact that injections of WGA-HRP centered caudal to the anterior commissure anterogradely labeled terminals at rostral but never at caudal levels of NTS. The observation that the rostral NTS receives a disproportion- ately heavier projection from the insular cortex was based on retrograde transport from injection sites occupying roughly similar volumes of NTS, but not encroaching on surrounding structures (such as the NPvc or spinal trigeminal nucleus), and was consistent with the overall higher density of cortically derived terminals at rostral vs. caudal levels of NTS. In addition, our control studies also demonstrated heavy terminal projections contiguous into the NTS from cortex to the parvicellular reticular formation. Neu- rons projecting to NPvc were, moreover, more broadly distributed and extended dorsally into the adjacent somatosensory area. In light of the above data it appears that topographic connections between cortex, NTS, and NPvc are not clear-cut and are more complexly organized than thought previously. Experiments using iontophoretic injections of tracers such as WGA-HRP into electrophysio- logically characterized sites, preferably at the level of the single cell, should, therefore continue to pursue the exact relationships between cortex and the individual subnuclei of NTS and the reticular formation.

Our anterograde transport data have also defined in greater detail than in previous reports (Saper, '82a; Shipley, '82; van der Kooy et al., '82) the precise distributions of cortically derived terminals within the NTS. In several areas, these patterns did not conform to the borders identified by Nissl stains or immunocytochemistry (Kalia and Sullivan, '82; Kalia et al., '84, '85).


Cortical projections to the dorsolateral field of the NTS, for example, were found to extend across the cytological boundaries of the dorsal, dorsolateral, and dorsal strip subnuclei as defined in the above studies. At levels caudal to the area postrema these labeled terminals formed, and thus identified, a new dorsal division of the commissural nucleus of the vagus.

Terminals in the ventral aspect of NTS revealed a new substructure adjacent to the dorsal motor nucleus that we have termed the ventromedial (arcuate) division. This division did not correspond to any of the recently reported subregions of the NTS as identified by Nissl staining and was clearly distinguished from the adjacent ventral subnucleus, which was more restricted in location and surrounded the ventral border of the solitary tract (Kalia and Sullivan, '82). Our ventromedial division, in contrast, con- formed to the arcuate contour of the dorsal motor nucleus throughout the medulla, and, on some tissue sections, al- most completely surrounded it. Retrograde transport experiments employing horseradish peroxidase conjugates have also revealed this structure on the basis of a similar arcuate pattern of cell bodies labeled from the hypothalamus (un- published data). The ventromedial subnucleus was also recognized on counterstained sections as a relatively loosely organized area of predominantly small to intermediate diameter, lightly stained neurons intervening between the medial division and the motor vagal nucleus. The conclu- sion that some of the cortically derived terminals in the ventromedial division may be ending on dendrites, perhaps of the motor nucleus and/or NTS, will require ultrastructural verification.

Cortical projections to the NTS also disclosed another, heretofore unidentified, field in the periventricular gray, subadjacent to the floor of the fourth ventricle. Termed the periventricular division, these terminals corresponded in counterstained sections to an array of elliptically shaped neurons running parallel to the ventricular floor. The nucleus is seen clearly in Figure 9A of Kalia and Sullivan ('82) but was not identified by them as a separate substructure. In more recent reports (Kalia et al., '84, '85) the general area was differentiated from the rest of the NTS, designated the periventricular region, and characterized by intense immunoreactivity to several neuropeptides and catecholamines and by dendrites of neurons in the adjacent NTS.

The insular cortex, by means of its widespread projections throughout the NTS, is likely able to modulate signals related to taste and a broad variety of visceral receptors, A more specific role in the integration of the baroreceptor reflex is suggested by the striking overlap between aortic nerve primary afferents as defined in rat (Ciriello, '83) and terminals labeled from the insular cortex. Convergence of both fields seems to occur in an area surrounding the tract and includes portions of the dorsolateral (as defined here), intermediate, ventrolateral, and commissural subnuclei. Based on om combined morphophysiological data, neurons in these divisions appear to represent an afferent limb of the baroreceptor reflex and project to an area of the rostral ventrolateral medulla essential for mediating vasodepressor responses and maintaining tonic vasomotor activity (Granata et al., '83, '85; Ross et al., '84a,b, '85). Subsequent studies should elaborate on the microcircuitry of these fields and the central integrating mechanisms mediating the cortical control of circulation.

205

Finally, the insular cortex may not be the only source of cortical autonomic fibers in the central nervous system; other cortical areas have also been reported in a variety of species to innervate brainstem autonomic nuclei and include medial and lateral parts of prefrontal cortex, the mediobasal surface and both motor and sensory cortical areas (Brodal et al., '56; Torvik, '56; Zimmerman et al., '64; Shriver and Noback, '67; van der Kooy et al., '82; Saper, '82b; Terreberry and Neafsey, '83). Wall and Davis ('51) identified three cortical systems separately mediating changes in arterial blood pressure: a corticospinal tract from the sensorimotor cortex, a temporopontine tract mediating temporal lobe and anterior cingulate autonomic function, and a corticohypothalamic pathway originating on the posterior orbital surface and within the anterior insular lobe. Descending projections from the motor cortex to thoracic autonomic neurons were also reported with degeneration techniques (Hoff and Hoff, '34; Smith et al., '67; Clarke et al., '68), although this connection has yet to be confrmed with axonal tracers. We have recently confirmed a small population of cells projecting to the NTS from deep layers of the infralimbic cortex as described by van der Kooy et al. ('82) and Terreberry and Neafsey ('831, although this area was rostral to the region investigated in this study. Preliminary data in rat have also recently shown changes in arterial pressure by stimulating the infralimbic cortex and most areas of the anterior cingulate and prelimbic cortex (Tucker and Saper, '84).

In keeping with the above anatomical data, the general consensus of most previous physiological investigations suggests that areas of cortical autonomic responsivity are more broadly dispersed than the insular pressor area described here and encompass nearly the anterior half of the cerebral hemisphere (Kennard, '45; Kaada, '51; Wall and Davis, '51; Delgado, '60). Although species differences and experimental parameters (depth and quality of anesthesia and stimulus characteristics) may account for conflicting results, there appears to be, as Delgado (1960) has empha- sized, a stability of cortical points from which specific autonomic responses can be elicited, consistently, and appa- rently independent of epileptiform activity, subcortical current spread, or meningeal stimulation. This view is strongly supported by the landmark work of Hoff and Green ('36) and Kaada ('51) and is further corroborated in this report by the tight correspondence between cortical pressor sites and an anatomically defined area of autonomic representation in insular cortex.

Subsequent studies will aim at a more complete systematic investigation of cerebral cortex in order to explore in greater depth the morphophysiological relationships between the insular pressor area and other areas of potential autonomic responsivity. It is not known, for example, whether the constellation of autonomic responses elicited from spatially separate and widespread areas of cortex, as reported previously, are transmitted independently or inte- grated by using a final common pathway.

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