THE JOURNAL OF COMPARATIVE NEUROLOGY 311~1-16 (1991)

Organization of Visceral and Limbic Connections in the Insular Cortex of the Rat GARY V. ALLEN, CLIFFORD B. SAF'ER, KAREN M. HURLEY, AND DAVID F. CECHETTO Department of Stroke and Aging, Robarts Research Institute, and Department of Physiology, University of Western Ontario, London, Ontario, Canada N6A 5K8 (G.V.A., D.F.C.), and Department of Pharmacology and Physiological Sciences, University of Chicago, Chicago, Illinois 60637 (K.M.H., C.B.S.)

ABSTRACT The anterograde and retrograde transport of horseradish peroxidase was used to study the anatomical organization of visceral and limbic terminal fields in the insular cortex. Following injections into the ventroposterolateral parvicellular (VPLpc) and ventroposteromedial parvicellular (VF'Mpc) visceral relay nuclei of the thalamus, dense anterograde and retrograde labeling was present in the posterior granular and dysgranular insular cortices, respectively. The parabrachial nucleus had extensive connections with the posterior dysgranular cortex and to a lesser degree with the anterior dysgranular and granular cortices. In contrast, injections into the medial prefrontal cortex and mediodorsal nucleus of the thalamus resulted in dense anterograde and retrograde labeling primarily in the anterior agranular cortex, whereas injections in the amygdala resulted in axonal labeling in the agranular and dysgranular insular cortices. Injections into the lateral hypothalamic area resulted in dense anterograde and retrograde labeling mainly in the agranular and dysgranular cortices and moderate to light labeling in the granular cortex. Our results indicate that ascending visceral afferents, VPLpc, WMpc, and parabrachial nuclei, are topographically organized in the granular and dysgranular fields of the insular cortex, whereas the agranular cortex appears to receive highly integrated limbic afferents from the infralimbic cortex and the mediodorsal nucleus of the thalamus. Although these visceral and limbic inputs to the insular cortex are segregated for the most part into different longitudinally oriented strips of cortex, limbic input from the lateral hypothalamic area and the amygdala, which have extensive autonomic as well as limbic connections, are more diffusely distributed over the different regions of the insular cortex. This organization may subserve a role for the insular cortex in integration of autonomic response with ongoing behaviour and emotion. Key words: limbic system, autonomic nervous system, cardiovascular,horseradish peroxidase

It has been suggested that the insular cortex represents a cortical region for the integration of limbic and autonomic responses (Saper, '82a). This concept has been supported in recent years by a number of investigations demonstrating that the insular cortex has connections with both visceral and limbic cell groups. Limbic structures such as the infralimbic cortex (Beckstead, '79; Saper, '82a; van der Kooy et al., '82; Hurley et al., 'go), amygdala (Krettek and Price, '77a; Saper, '82a), and medial dorsal nucleus of the thalamus (Krettek and Price, '77b; Saper, '82a) are extensively interconnected with the insular cortex. Visceral information from the nucleus of the solitary tract is relayed to the insular cortex sequentially through the parabrachial Q

1991 WILEY-LISS, INC.

nucleus and the ventral basal thalamus in a topographically organized manner (Cechetto and Saper, '87, '90). These investigations have shown that the ventroposteromedial parvicellular (WMpc) and ventroposterolateral parvicellular (VPLpc) nuclei of the thalamus are likely relay sites for gustatory and general visceral afferent information, respectively. In addition, the parabrachial nucleus projects directly to the insular cortex (Saper, '82a,b). The lateral Accepted May 17,1991. Address reprint requests to Dr. David F. Cechetto, John P. Robarts Research Institute, 100 Perth Dr., P.O. Box 5015, London, Ontario, Canada N6A 5K8.

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2 hypothalamic area, which is the primary relay for sympathetic responses originating in the insular cortex (Cechetto and Chen, 'go), is also reciprocally connected to the insular cortex (Saper, '82a). Although there is some evidence for an anterior-posterior organization of ventral basal thalamic inputs to the insular cortex (Cechetto and Saper, '87), the anatomical relationship of visceral versus limbic connections in the insular cortex is not known. We studied the organization of afferents to the insular cortex by using injections of horseradish peroxidase (HRP) and HRP conjugated to wheat-germ agglutinin (WGA-HRP)into the medial prefrontal cortex, amygdala, thalamus, lateral hypothalamic area, and parabrachial nucleus.

MATERIALS AND METHODS In 43 rats, pressure injections of a 1%aqueous solution of WGA-HRP (5-10 nl) or iontophoretic injections (2-6 FA positive, 7 seconds on and 7 seconds off cycle for 5-10 min) of a 1%solution of HRP (Sigma TYPE VI) or 2% solution of WGA-HRP (Sigma) in 0.1 M phosphate-buffered saline (pH 7.3) were made. Of these injections, 7 were made in the infralimbic area, 4 in the amygdala, 6 in the medial dorsal nucleus, 8 in the VPMpc, 8 in the VPLpc, 3 in the lateral hypothalamic area, and 7 in the parabrachial nucleus. Injections were placed stereotaxically under chloral hydrate anesthesia (350 mg/kg i.p.) by using either a glass micropipette syringe for the pressure injections (Saper, '83) or microfilament glass electrodes and a constant current source (Edco Scientific Inc.) for the iontophoretic injections. After 1-3 days, the animals were reanesthetized with chloral hydrate and perfused through the heart with saline followed by a 0.5%paraformaldehyde 1.25%glutaraldehyde solution in 0.1 M phosphate buffer (pH 7.3). The brains were removed and 50-pm sections were cut on a freezing microtome and processed by the tetramethyl benzidine method of de Olmos et al. ('78) or Mesulam ('78). Sections were mounted on gelatin-coated glass slides and stained with a 0.1%thionin solution.

Abbreviations A

ac A1 AMYG

AON CLA DI

E EN GI i-vi IL

LF LHA M MD OLB PB PIR Prh

SI SII V VPLpc VPMpc

auditory cortex anterior comrnissure agranular insular cortex amygdala anterior olfactory nucleus claustrum dysgranular insular cortex entorhinal cortex endopiriform nucleus granular insular cortex cortical layers infralimbic cortex lateral frontal cortex lateral hypothalamic area motor cortex mediodorsal nucleus of the thalamus olfactory bulb parabrachial nucleus piriform cortex perirhinal cortex somatosensory cortex, area I somatosensory cortex, area I1 visual cortex ventroposterolateral parvicellular thalamic nucleus ventroposteromedialparvicellular thalarnic nucleus

Sections were viewed and photographed with brightfield or combined polarized-darkfield optics. The locations of labeled cell bodies and terminal fields were mapped using a camera lucida drawing tube. Fine punctate labeling was taken to indicate terminal labeling in the insular cortex, which represented the endpoint of the efferent pathway. Labeling observed medial to the external capsule was not plotted in the camera lucida drawings. The lateral view of the rat cerebral cortex illustrated in Figure 10 is a modification of that produced by Zilles et al. ('80).

RESULTS Following injections of HRP or WGA-HRP into visceral and limbic areas, dense anterograde and retrograde labeling was observed in the granular, dysgranular, or agranular insular cortices. These three longitudinally oriented regions of the insular cortex have been identified on the basis of cytoarchitecture and neural connections (Zilleset al., '80; Cechetto and Saper, '87). The insular cortex is defined as the region of cortex overlyingthe claustrum that is bounded rostrodorsally by the lateral frontal cortex and somatosensory cortex SI and caudodorsallyby SII (Fig. 1).The insular cortex is bounded ventrally by the anterior olfactory nucleus and piriform cortex and caudally by the perirhinal cortex (see summary Fig. 10). The granular insular cortex, immediately ventral to the somatosensory areas SI and SII, is characterized by a well-developed granular layer IV. Ventral to the granular cortex is an intermediate strip of dysgranular cortex in which granule cells in layer IV are markedly diminished in numbers and layer V is more prominent. The dysgranular insular cortex extends anterior to the granular insular cortex and is found ventral to the lateral frontal cortex. The most ventral region, the agranular insular cortex, is located primarily in the groove of the rhinal fissure. The agranular cortex extends rostrally into the anterior portion of the frontal pole. The agranular insular cortex is characterized by three prominent cell layers: layers 11-111, V, and VI. The granular, dysgranular, and agranular insular cortices end caudally just anterior to the perirhinal cortex. Figure 1 demonstrates the distinctive organization of granular, dysgranular, and agranular cortical regions in three evenly spaced rostral to caudal levels of the normal insular cortex. These representative levels are used throughout the results to illustrate the pattern of anterograde and retrograde labeling in the insular cortex.

Ventroposterolateral parvicellular nucleus of the thalamus Following injections of tracer into the VPLpc (Fig. 2A), dense anterograde labeling was observed almost exclusively in the middle and caudal portions of the granular insular cortex (Figs. 2B, 3A, see also Fig. 10A). In case R302 (Fig. 2A), the injection site included the VPLpc with light spread of tracer dorsally into the ventroposterolateral nucleus and ventrally into the dorsolateral part of the zona incerta, which do not project to the insular cortex (Saper, '82a). Labeled fibers extended laterally from the injection site into the internal capsule, traversed the striatum in a dorsolatera1 direction, entering the external capsule, and terminating in the insular cortex, ipsilaterally. In the granular insular cortex (Figs. 2B, 3A), dense anterograde labeling was observed in layers 111, IV, and VI. Moderate to light anterograde labeling was observed in layers I1 and V. Dense

Fig. 1. Photomicrographs of thionin-stained coronal sections through the lateral frontal cortex showing the cytoarchitectonic organization of the insular cortex. The sections have been taken from the middle of each rostral-caudal third of the insular cortex. The dashed lines indicate the boundaries of the granular (GI), dysgranular (DI), and agranular (AI) cortical areas. A is anterior and C is posterior. Scale bar: 500 km.

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Fig. 2. Photomicrographs of WGA-HRP injection sites in the thalamus and resultant labeling in the insular cortex. A. Injection site in the VPLpc (R302). B. Anterograde and retrograde labeling in the granular insular cortex followingthe injection shown in A. C. Injection site in the

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WMpc (R304)and resultant anterograde and retrograde labeling in the dysgranular insular cortex (D). (cf. description of the injection in Case R304 in Cechetto and Saper, '87.) Scale bars: 500 km.

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VPMpc.

Fig. 3. Camera lucida drawings of coronal sections of the insular cortex showing the distribution of anterograde (fine stippling) and retrograde labeling (large dots represent 1-4 labeled cells) following injections of tracer into the VPLpc (R302) (A) and the VPMpc (R304)(B).The sections in A and B are arranged in order from anterior to posterior and correspond with the anterior-posterior levels shown in Figure 1.Scale bar: 1 mm.

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Fig. 4. Photomicrograph of WGA-HRP injection site in the parabrachial nucleus (R663) (A) and resultant anterograde and retrograde labeling in the insular cortex (B).Arrowheads indicate the border of the dense uniform label that demarcates the core of the injection site. Scale bars: 500 km.

retrograde labeling was located mainly in layer VI and the deep part of layer V.

Ventroposteromedial parvicellular nucleus of the thalamus Figure 2C shows a representative injection (R304) into the VF'Mpc. There was some spread of tracer from the core of the injection site dorsally and medially into the intralaminar thalamic nuclei, including the parafascicular and centromedial nuclei. The parafascicular thalamic nucleus does not project to the insular cortex (Saper, '82). Although the centromedial nucleus is known to project to the insular cortex (Saper, 'BZa), the pattern of labeling in the insular cortex illustrated in case R304 occurs only when the VPMpc is included in the central core of the injection site. In addition, analysis of a number of cases in this study indicates that very little or no detectable transport of HRP or WGA-HRP occurs from the spread zone of the injection site. Labeled fibers projected laterally from the injection site on the dorsal surface of the medial lemniscus into the internal capsule, then passed laterally through the striatum to reach the dysgranular portion of the insular cortex, ipsilaterally (Figs. 2D, 3B; see also Fig. 10B). Anterograde and retrograde labeling was concentrated throughout the dysgranular insular cortex except for the anteriormost portion and extended dorsally into the ventral portion of the granular insular cortex (Fig. 3B; see also Fig. 10B). Dense anterograde labeling was located in layers I, 111, IV, and the superficial parts of layer V of the dysgranular insular cortex. Moderate to light anterograde labeling was

observed in layer I1 and the deep portion of layer V and layer VI. Heavy retrograde labeling was observed in layer VI and moderate to light retrograde labeling was observed in a superficial region of layer V of the dysgranular cortex.

Parabrachial nucleus Following injections into the parabrachial nucleus, dense anterograde labeling was located in the dysgranular cortex (Figs. 4B, 5; see also Fig. 10B). Case R663 shows the distribution of labeling in the insular cortex following a large injection into the parabrachial nucleus. The core of the injection site in case R663 (Fig. 4A) involved both medial and lateral parts of the parabrachial nucleus. The halo but not the central core of the injection site involved the Kolliker-Fuse nucleus, motor trigeminal nucleus, and mesencephalic trigeminal nucleus, locus coeruleus, and laterodorsal tegmental nucleus. Analysis of a number of cases with small injections into this region indicated that very little or no detectable transport of tracer occurs from the spread zone of the injection site. The distribution of parabrachial fibers and terminals in the forebrain has been described extensively elsewhere (Saper and Loewy, '80; Fulwiler and Saper, '84; Cechetto and Saper, '90) and is not detailed here. Labeled axons from the injection site ascended in the central tegmental tract to the diencephalon where they continued rostrally ventral to the medial lemniscus and medial to the zona incerta in the medial forebrain bundle. Corticopetal s o n s ran laterally from the lateral hypothalamic area, through the ansa peduncularis, traversing the amygdala and ventral stria-

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Fig. 5. Camera lucida drawings of coronal sections through the insular cortex showing the distribution of anterograde (fine stippling) and retrograde (large dots represent 1-4 labeled cells) labeling following injection of tracer into the parabrachial nucleus (R663).The

sections are arranged in order from anterior to posterior and correspond with the anterior-posterior levels shown in Figure 1. Scale bar: 1mm.

tum to reach the external capsule and the insular cortex, predominately ipsilaterally. Anterograde and retrograde labeling was concentrated throughout the dysgranular insular cortex except for the anteriormost portion and extended dorsally into the ventral portion of the granular insular cortex (Fig. 5; see also Fig. 10B). The densest concentration of labeling was present diffusely in layers I through VI of the caudal portion of the posterior dysgranular insular cortex and extending dorsally into the ventral region of the granular insular cortex and, to a lesser degree, ventrally into the posterior agranular insular cortex. Moderate retrograde labeling was located mainly in layer V of the dysgranular insular cortex and a few labeled cells were located in layer V of the adjacent granular and agranular insular cortices (Fig. 5 ) .

forebrain bundle via the ansa peduncularis into basal forebrain structures such as the ventral pallidum, ventrolateral caudate-putamen and substantia innominata before reaching the external capsule and insular cortex, predominately ipsilaterally. In the amygdala, dense anterograde labeling was seen in the central, lateral, basolateral, basomedial, and medial nuclei, and heavy retrograde labeling was seen in the central, basomedial and anterior cortical nuclei. In the insular cortex, anterograde and retrograde labeling was observed in the granular, dysgranular, and agranular regions of the insular cortex (Fig. 7 4 see also Fig. lOC). Terminal labeling was most dense in layers V and VI of the agranular and dysgranular insular cortices with moderate to light terminal labeling extending into the adjacent granular insular cortex. Light anterograde labeling was present in layers 11-111 of all three insular cortical regions (Figs. 6B, 7A). Light to moderate anterograde labeling was also widely distributed in somatosensory areas I and 11, the claustrum, endopiriform nucleus, and the piriform cortex. Large numbers of retrogradely labeled cells were present in layer V of the dysgranular and agranular insular cortices, but only a few retrogradely labeled cells were seen in layer VI of the dysgranular and agranular cortices, the claustrum, and layer V of the granular insular cortex.

Lateral hypothalamic area Following injections of tracer into the lateral hypothalamic area (Fig. 6A, case RC10) extensive anterograde and retrograde labeling was seen in the insular cortex (Figs. 6B, 7A). The core of the injection site shown in Figure 6A was centered in the posterior region of the lateral hypothalamus and involved tuberal and posterior portions of the lateral hypothalamic area. There was some spread of tracer from the injection site dorsally into the zona incerta and laterally into the subthalamic nucleus. Labeled fibers left the injection site and projected rostrally in the medial forebrain bundle. Labeled fibers extended laterally from the medial

Amygdala Following injections of tracer into the lateral and basolateral nuclei of the amygdala (Fig. 6C, case RC9), labeled

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Figure 6

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9

fibers ascended rostrally and dorsally into the external capsule to terminate in the insular cortex. The core of the injection site extended medially into the central nucleus of the amygdala and dorsally into the striatum, but these sites do not project to the insular cortex (Saper, '82a). Dense anterograde and retrograde labeling was observed in the agranular insular cortex except for the anteriormost portion and in the dysgranular insular cortex except for the caudalmost portion (see Fig. 10D). In the agranular and adjacent dysgranular insular cortices (Figs. 6D, 7B), dense anterograde labeling was distributed over layers I, 11-111, V, and VI and retrogradely labeled cell bodies were located mainly in layers 11-111 and V. The terminal labeling was more dense in the anterior insular cortex compared to the posterior insular cortex. Light anterograde and retrograde labeling was also present in the contralateral agranular and dysgranular areas following injection of tracer into the amygdala.

nucleus to the internal capsule. Labeled fibers passed rostrally in the internal capsule and extended laterally through the caudate-putamen to the external capsule and into the insular cortex. Anterograde and retrograde labeling was observed in the rostrocaudal agranular insular cortex and in the adjacent intermediate portion of the dysgranular insular cortex (Figs. 9B, 10F). Rostrally, dense anterograde labeling was observed in layers I, 111, V, and VI of the agranular insular cortex and the adjacent portion of the dysgranular insular cortex (Figs. 8D, 9B). In the posterior insular cortex the terminal labeling was much lighter and restricted to layers V and VI of the agranular insular cortex. Large numbers of retrogradely labeled cells were located in layers V and VI of the rostral agranular and adjacent dysgranular insular cortices. Contralaterally, moderate to light retrograde labeling was present in layer VI of the agranular and adjacent dysgranular insular cortices.

Medial prefrontal cortex

DISCUSSION

Figure 8A shows a representative case (R441) of an injection of tracer into the infralimbic area. There was some spread of tracer dorsally into the prelimbic area and ventrally into the dorsal peduncular cortex, but neither of these sites project to the insular cortex (Hurley et al., submitted). The labeled fibers from the injection site reached the insular cortex by projecting caudally for a short distance before crossing laterally in the ventral caudate-putamen to reach the external capsule and the insular cortex (Hurley et al., submitted, has a more complete description of this pathway). Anterograde and retrograde labeling was observed primarily in the anterior portion of the agranular insular cortex and the adjacent ventral portion of the dysgranular insular cortex (see Figs. 9A, 10E). Terminal labeling was most dense in rostral and intermediate levels of the agranular insular cortex and the adjacent portion of the dysgranular insular cortex, with lighter labeling at more caudal levels of the agranular insular cortex (Figs. 8B, 9A). Rostrally, anterograde labeling was observed throughout layers I-VI, whereas retrograde labeling was seen in layers 11-111 and VI as well as the underlying claustrum. The superficial labeling was not observed in the posterior insular cortex, where the anterograde and retrograde labeling was restricted to layer VI. Very light anterograde and retrograde labeling was also present in similar regions of the contralateral agranular insular cortex.

The central focus of this work is a study of the sources and relative distributions of highly organized specific inputs to the insular cortex. For this study, HRP and WGA-HRP were used to trace insular cortical connections for the following reasons. First, the HRP and WGA-HRP injections usually encompassedthe rostrocaudal and mediolateral extent of structures being studied. This was advantageous because we are interested in examining the complete projection from structures, whereas other anterograde tracers such as Phaseolus vulgaris-leucoagglutinin produce small injection sites which are more appropriate for studying the subnuclear organization of a particular structure. Second, the retrograde labeling seen with the peroxidase tract tracing technique provides important information regarding the number and distribution of neurons in the insular cortex and other sites with efferent projections to injection sites. Third, with the small volumes and low concentrations of HRP and WGA-HRP used in this study, the degree of labeling arising from diffuse cortical projecting structures, such as the intralaminar thalamic nuclei, locus coeruleus, and the raphe nuclei (see Saper, '87), was minimal. The organization of diffuse, highly collateralized inputs from structures of diffuse cortical projection systems has been addressed in previous reports (for review see Saper, '87). Our results indicate that the insular cortex (as defined by Rose, '281, receives topographically organized afferents Mediodorsal nucleus of the thalamus from a number of major visceral and limbic sites in the rat The injection site in Figure 8C (case R579) is centered in brain. Figure 10 summarizes the inputs only to the insular the mediodorsal nucleus of the thalamus. The injection cortex from limbic and visceral sites. The organization of included parts of the paraventricular and intermediodorsal these terminal fields into longitudinally oriented strips thalamic nuclei medially, and the stria medullaris of the primarily restricted to one or more of the granular, dysgranthalamus and the habenular nucleus dorsally, but none of ular, or agranular fields suggests that the insular cortex these additional sites project to the insular cortex (Saper, may be important for the integration and regulation of '82a). Labeled fibers were observed projecting from the autonomic and limbic system activity since interconnecinjection site ventrolaterally through the reticular thalamic tions between the different regions of the insular cortex have been indicated Cyasui et al., '91). Pronounced differences in the pattern of connectivity between infralimbic cortex and mediodorsal nucleus of the Fig. 6. Photomicrographs of WGA-HRP injection sites in the lateral thalamus and the VPMpc, VPLpc and parabrachial nuclei hypothalamic area (RC10) and amygdala (RC9) and resultant labeling were observed. The former afferents terminated primarily in the insular cortex. A. Injection site in the lateral hypothalamic area. B. Anterograde and retrograde labeling in the insular cortex. C . in the agranular insular cortex, particularly anteriorly, whereas the latter afferents mainly innervated the granular Injection site in the lateral, basolateral, and central nuclei of the and dysgranular insular areas. In contrast, the amygdala amygdala. D. Resultant anterograde and retrograde labeling in the and lateral hypothalamic area showed more extensive agranular and dysgranular insular cortices. Scale bars: 500 Vm.

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Fig. 7. Camera lucida drawings of coronal sections of the insular cortex showing the distribution of anterograde (fine stippling) and retrograde (large dots represent 1-4 labeled cells) labeling following injections of tracer into (A) the lateral hypothalamic area (LHA, case

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RC10) and (B)amygdala (AMYG, case RC9). The sections in A and B are arranged in order from anterior to posterior and correspond with the anterior-posterior levels shown in Figure 1.Scale bar: 1mm.

VISCERAL AND LIMBIC ORGANIZATION OF INSULAR CORTEX

Fig. 8. Photomicrographs of WGA-HRP injection sites in the medial prefrontal cortex (R441) and the thalamus (R579) and resultant labeling in the insular cortex. A. Injection site centered in the infralimbic area. B. Resultant anterograde and retrograde labeling in the

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agranular and dysgranular insular cortices. C. Injection site in the mediodorsal nucleus of the thalamus and resultant anterograde and retrograde labeling in the agranular area and ventral portion of the dysgranular area (D).Scale bars: 500 km.

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Fig. 9. Camera lucida drawings of coronal sections of the insular cortex showing the distribution of anterograde (fine stippling) and retrograde (large dots represent 1 4 labeled cells) labeling following injections of tracer into (A) the infralimbic cortex (IL, case R441) and

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(B)mediodorsal nucleus of the thalamus (MD, case R579). The sections in A and B are arranged in order from anterior to posterior and correspond with the anterior-posterior levels shown in Figure 1. Scale bar: 1mm.

VISCERAL AND LIMBIC ORGANIZATION OF INSULAR CORTEX 1.6

0.6

I

I

-0.4 I

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-1.4 Bregma I

Fig. 10. Line drawings of the lateral view of the rat cerehral cortex showing the distrihution of inputs (diagonal lines) only to the insular cortex from visceral (WLpc, WMpc and PB) and limbic (LHA, AMYG, 1L and MD) sources. The diagonal lines show only the extent of the input to the insular cortex and does not distinguish between areas receiving different intensities of labeling.

insular connections. The lateral and basolateral nuclei of the amygdala project to both the anterior and posterior agranular and dysgranular insular cortices. The lateral hypothalamic area was connected with all three regions of the anterior and posterior insular cortex. This suggests that the afferents from the amygdala and the lateral hypothalamic area may influence the processing of input from various sources in the insular cortex. This role is consistent with the well-known importance of the amygdala and hypothalamus in autonomic regulation as well as behaviour and emotion (Anand and Brobeck, '51; Kaada, '51; Smith,

'56; Fernandez de Molina and Hunsperger, '59; Folkow and Rubinstein, '65; Epstein, '71; Opsahl, '77; Grijalva, '80; Kapp et al., '81; Siege1 and Edinger, '81; Stock et al., '81; Cechetto and Calaresu, '83, '84).

Visceral connections of the insular cortex The present study confirms and extends previous investigations of the afferent and efferent connections of the insular cortex. The parvicellular thalamic relay nuclei (VPLpc and VPMpc) have topographically organized projec-

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context to autonomic responses originating in the agranular insular cortex (Yasui et al., '91). Our data confirm the results of previous studies that have demonstrated ascending projections from the mediodorsal nucleus of the thalamus to the insular cortex (Krettek and Price, '77b; Saper, '82a). In addition, the present results confirm that cells in layers V and VI of the agranular insular cortex and adjacent dysgranular cortex project to the mediodorsal nucleus (Kosar et al., '86a,b; Groenewegen, '88; Krushel and van der Kooy, '88). The mediodorsal nucleus also receives inputs from the medial prefrontal cortex (Beckstead, '761, ventral pallidum (Haber et al., ,851, amygdala (Krettek and Price, '77a) hippocampal formation (Irle et al., '84), lateral hypothalamus (Hosoya and Matsushita, '81), central gray (Eberhart et al., '851, median raphe nucleus (Azmitia and Segal, '781, and reticular formation (Siege1 et al., '77). Thus many of the brain sites with direct projections to the insular cortex may also influence the insular cortex following processing in the mediodorsal nucleus of the thalamus. In contrast to the region-specificinputs from the infralimbic cortex and the thalamic mediodorsal nucleus, projections from the lateral hypothalamus and the amygdala are distributed over two or more regions of the insular cortex (see summary Fig. 10). The lateral hypothalamic area has reciprocal connections with the agranular and dysgranular insular cortices, as well as some overlap into the granular region. The sites for cardiovascular and gastrointestinal responses following electrical and chemical stimulation of the insular cortex (Yasui et al., '91) are very similarly distributed. Furthermore, the lateral hypothalamic area is a mandatory relay for sympathetic responses originating in the insular cortex (Cechetto and Chen, '90). Hence, insular connections with the lateral hypothalamic area may provide the anatomical substrate not only to mediate autonomic responses, but also to provide feedback to the insular cortex. Afferent projections from the amygdala arise from the lateral and basolateral nuclei (Krettek and Price, '77a; Limbic connections of the insular cortex Saper, '82a) and terminate in layers I-VI of the agranular Previous studies indicated that the insular cortex re- and dysgranular insular cortices. In addition, neurons in ceives projections from the medial prefrontal cortex (Beck- agranular and dysgranular regions project to the basolatstead, '79; Saper, '82a; van der Kooy et al., '82) and the eral, lateral, and central nuclei of the amygdala (Veening, medial portion of the mediodorsal nucleus of the thalamus '78; Ottersen, '82; Saper, '82a). Ottersen ('82) indicated (Krettek and Price, '77b; Saper, '82a). However, little was that cells in layer V of the insular cortex project to the known of the anatomical relationships of the terminal field central nucleus and cells in layers 11-111 project to the in the insular cortex. Using the anterograde and retrograde basolateral and lateral nuclei. Recent electrophysiological transport of HRP or WGA-HRP, we have demonstrated studies have recorded short and long latency excitatory and that afferents from the above sites converge in the agranu- inhibitory responses in the central nucleus of the amygdala lar insular cortex with slight overlap into adjacent portions following stimulation of the insular cortex in the rat and of the dysgranular cortex. rabbit (Yamamoto et al., '84; Pascoe and Kapp, '87). Since In the medial prefrontal cortex, efferent fibers arise the lateral hypothalamic area and the amygdala have dense primarily from neurons in the layer I11 of the infralimbic connections with both limbic and autonomic structures, the region (Saper, '82a; Hurley et al., '90) and project to the distribution of these inputs over several regions of the agranular insular cortex (Hurley et al., '90; present re- insular cortex may subserve a role in integration of autosults). The layer I11 of the infralimbic cortex also projects to nomic responses with ongoing behavior and emotion. a number of forebrain sites related to autonomic function, including the lateral hypothalamic area, central nucleus of Functional considerations the amygdala, and bed nucleus of the stria terminalis (Hurley et al., '90). The relationships of neurons projecting The presence of projections from the insular cortex to to these different targets are not known. various limbic and visceral sites suggests that the insular As the infralimbic cortex receives afferents from a num- cortex may play a role in visceromotor and behavioral ber of limbic structures such as CA1 field of hippocampus, responses (Krushel and van der Kooy, '88; present results). basolateral amygdala, and prelimbic cortex (Tucker and These results have implications for both health and disease. Saper, '85), it may provide a behavioral and emotional Recent investigations have demonstrated that cardiovascu-

tions to the insular cortex (Cechetto and Saper, '87, present results). The VPLpc and WMpc project to separate but adjacent terminal fields in the granular and dysgranular insular cortices, respectively. The WMpc relays gustatory information from the rostral portion of the nucleus of the solitary tract to the insular cortex (Ganchrow and Erickson, '72; Norgren and Wolf, '75; Cechetto and Saper, '87). In contrast, the VPLpc receives general visceral information relayed by the parabrachial nucleus from the posterior region of the nucleus of the solitary tract (Roberts and Akert, '63; Cechetto and Saper, '87, '90; Herbert and Saper, '90). Rogers et al. ('79) demonstrated that a band of cells (probably in the VPLpc) located lateral to the VPMpc responded to infusion of small amounts of sodium chloride into the hepatic portal vein. More recently, Cechetto and Saper ('90) demonstrated that changes in the activity of single unit recording of neurons in the WLpc were made in response to activation of arterial baroreceptors and chemoreceptors, and gastric mechanoreceptors. The general visceral region of the thalamus projects to the granular insular cortex. Within the general visceral cortex, responses to cardiopulmonary stimulation can be elicited from neurons in caudal portions, whereas gastric mechanoreceptor activation induces changes in the activity of neurons located more rostrally. This viscerotopic organization is maintained throughout the neuraxis (see Cechetto and Saper, '90 for review). In addition, the insular cortex receives direct projections from the parabrachial nucleus (Saper, '82a,b; Shipley and Sanders, '82). The present study shows that afferent fibers from the parabrachial nucleus terminate primarily in the caudal dysgranular and ventral granular cortex. It is not clear what type of information is conveyed by these fibers. However, the continuation of the parabrachial projection into the lateral frontal cortex suggests that it may serve a more generalized role such as arousal rather than a discrete sensory function.

VISCERAL AND LIMBIC ORGANIZATION OF INSULAR CORTEX lar responses can be elicited from discrete regions of the agranular insular cortex (Ruggiero et al., ’87; Yasui et al., ’91; Oppenheimer and Cechetto, ’90a). Furthermore, electrocardiographic changes associated with stroke may occur as a result of damage to the insular cortex (Cechetto et al., ’89, submitted). In fact, arrhythmias leading to asytole can be induced by stimulation of the insular cortex coincident with the QRS complex (Oppenheimer et al., ’90b). Previous studies have demonstrated a viscerotopic sensory representation in the insular cortex (Cechetto and Saper, ’87). A gustatory region was located in the anterior dysgranular insular cortex, whereas a general visceral sensory region was located in the granular insular cortex. Within the granular region, gastric mechanoreceptorresponsive units were situated in the anterior granular region, whereas cardiorespiratory-responsive units were situated more ventrally and posteriorly. The gustatory region of the insular cortex is believed to play a role in discriminating taste quality (Campbell, ’05; Benjamin and Akert, ’59; Grill and Norgren, ’781, including conditioned taste aversion (Yamamoto et al., ’80, ’81;Lasiter et al., ’85). Autonomic responses associated with insular lesions have received little attention, although Kennard (’45) demonstrated visceromotor and somatomotor changes typical of “sham rage’’ (Cannon and Britton, ’25) in cats following lesions including the insular cortex. In summary, our observations support the view that the granular and dysgranular fields mainly receive visceral afferents, whereas limbic afferents terminate largely in the agranular insular cortex. Reciprocal projections from the granular and dysgranular areas innervate visceral sensory nuclei, but descending projections to sites mediating visceromotor and behavioral responses (i.e., hypothalamus and amygdala)originate from granular, dysgranular, and agranular areas. This schema suggests that local cortico-cortical connections between the insular cortical fields may be important for the integration of visceral sensation with ongoing behaviour and autonomic response.

ACKNOWLEDGMENTS This research was supported by N522835 and American Heart Association-Wyeth-AyerstGrant-in-aid 881120 (CBS) and HSFO B.1417 (D.F.C.).D.F.C. is a recipient of a Heart and Stroke Foundation of Canada Scholarship.

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Organization of visceral and limbic connections in the insular cortex of the rat.

The anterograde and retrograde transport of horseradish peroxidase was used to study the anatomical organization of visceral and limbic terminal field...
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