The efferent connections to the thalamus and brainstem of the physiologically defined eye field in the rat medial frontal cortex

Brain Research Bulletin, Vol. 54, No. 2, pp. 175–186, 2001 Copyright © 2001 Elsevier Science Inc. Printed in the USA. All rights reserved 0361-9230/01/$–see front matter

PII S0361-9230(00)00444-5

The efferent connections to the thalamus and brainstem of the physiologically defined eye field in the rat medial frontal cortex Paola Guandalini* Dipartimento di Scienze Biomediche e Terapie Avanzate, Sezione di Fisiologia umana, Universita` di Ferrara, Ferrara, Italy [Received 27 April 2000; Revised 27 September 2000; Accepted 27 October 2000] ABSTRACT: The anterograde tracer Phaseolus vulgaris leucoagglutinin (PHA-L) was injected into sites of the rat frontal eye field (FEF) located in the medial frontal cortex. After a single iontophoretic injection of PHA-L into a FEF site where intracortical microstimulation elicited eye movements, anterogradely labelled fibres and terminal-like elements were found in the thalamus in the anterior nuclei, intralaminar nuclei, lateral portion of the mediodorsal nucleus and posterior nuclear group. In the midbrain and pons, labelled fibres were located in the anterior pretectal area, Darkschewitsch nucleus, superior colliculus and dorsolateral portion of the central gray. When the tracer was injected at the FEF periphery, at a site the stimulation of which evoked both eye and whisker movements, labelling distribution in the thalamus differed from that observed after FEF injections, while a similar distribution was observed in the brainstem. In the thalamus, anterograde labelling was observed in these latter cases in the anterior nuclei, ventral nuclei, medial portion of the laterodorsal nucleus. The present findings point out that the FEF and FEF periphery are connected with numerous subcortical structures of the thalamus and brainstem. In addition, the connections of FEF and FEF periphery with the thalamus differ, whereas the midbrain and pons connections of the two subdivisions share common targets. © 2001 Elsevier Science Inc.

projections of this area with the thalamus and brainstem [29,55]. However, similar studies have not previously been performed in the rat. Although the organisation of subcortical projections of the medial frontal cortex in the rat has been investigated in previous works [10,27,43,46], FEF was not electrophysiologically identified in these studies. To provide further clues on the role of FEF in the rat cortex, the cortical projections to the thalamus and brainstem of the rat FEF were investigated in the present study by means of Phaseolus vulgaris leucoagglutinin (PHA-L) tract tracing. The tracer was delivered within cortical sites where intracortical microstimulation (ICMS) evoked eye movements. MATERIALS AND METHODS The experiments were carried out on a total of nine male Wistar rats (250 –300 g). Details of the general methods of animal preparation, stimulation, and histological procedures have been described elsewhere [23]. Adequate measures were taken to minimize pain or discomfort to animals and care and handling of animals were approved by the Animal Research Committee of the University of Ferrara, in accordance with the National Institutes of Health guidelines. The animals were anaesthetised with ketamine hydrochloride (100 mg/kg, intraperitoneal [i.p.]) and placed in a stereotaxic frame. Craniotomy was performed from 3 mm anterior to 3 mm posterior to the bregma, and from the midline to 3 mm laterally [41]. A piezoelectric micromanipulator was used to position and advance a glass-insulated tungsten microelectrode (10 –20-␮m exposed tip and 0.6 –1.0 M⍀ resistance). ICMS was delivered by a Grass S88 stimulator with a constant current isolation unit (Grass PSIU6, Grass Instruments, Quincy, MA, USA). Trains (300 ms) of high-frequency (350 Hz) cathodal pulses of 0.25 ms duration were used throughout the experiment [40]. The stimulus intensity did not exceed 50 ␮A, and ICMS was made at 100 ␮m steps at a depth ranging from 1.0 to 1.7 mm from the pial surface. Minimum threshold values were found at a depth of 1.5 mm. One to six penetrations per animal were performed avoiding tissue damage; the ICMS was delivered at a depth of 1.5 mm from the pial surface. In seven rats, once a cortical site was found to elicit eye move-

KEY WORDS: Frontal eye field, Cortical projections, Phaseolus vulgaris leucoagglutinin, somatomotor cortex.

INTRODUCTION The frontal eye field (FEF) of primates plays a central role in the control of voluntary eye movements [7,47,53], as well as in the coordination of eye and head movements [6,57]. In the rat, the thin strip of the medial frontal cortex lying near the interhemispheric fissure and bordering the vibrissae area of the somatomotor cortex is considered homologous to the primate FEF [16,19,24,35,40,44,46]. However, a previous study of the corticocortical projections of the rat FEF indicated that in this species the FEF is implicated in the control of orienting and exploring behaviours in addition to its role in the control of saccadic eye movements [23]. Studies on the subcortical efferents of FEF in primates have pointed out substantial and discrete

* Address for correspondence: Dr. Paola Guandalini, Dipartimento di Scienze Biomediche e Terapie, Avanzate, Sezione di Fisiologia umana, Universita` di Ferrara, Via Fossato di Mortara, 17/19, I-44100 Ferrara, Italy. Fax: ⫹532 291242; E-mail: [email protected]

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FIG. 1. Schematic location on the cortex surface of the 37 penetrations performed. Numbers in A–P are distances in millimetres relative to bregma. R1–9 indicate the nine injected Phaseolus vulgaris leucoagglutinin sites.

ments by ICMS, the microelectrode was withdrawn and replaced with a glass micropipette (tip external diameter: 15–30 ␮m) filled with PHA-L (Vector Lab, Burlingame, CA, USA; 25% in 0.05 M phosphate buffer, pH 7.4) [18]. The micropipette tip was lowered, under stereotaxic and stereomicroscopic controls, into the same cortical site and at the same depth. The tracer was then injected by applying a constant positive current of 5 ␮A for 20 min. Each animal received a single iontophoretic PHA-L injection. In the remaining two animals (cases R7 and R9), PHA-L was injected at the periphery of the FEF, at the border between FEF and whisker motor area representation the ICMS of which evoked both eye and whisker movements; 250 ␮m laterally to this site ICMS evoked whisker movements only. Twelve to 17 days after the PHA-L injection, the animals were deeply anaesthetised with ketamine hydrochloride (100 mg/kg, i.p.) and perfused through the ascending aorta with heparinized phosphate-buffered saline followed by a mixture of 4% paraformaldehyde and 10% saturated picric acid in phosphate buffer (PB 0.1 M; pH 7.4). The brains were removed, postfixed for 8 –12 h in the same fixative and then transferred to a 30% sucrose solution PB until they sank. They were then sectioned coronally into 25–50-␮m-thick sections with a freezing microtome. Four series of adjacent sections were collected in cold potassium phosphate-buffered saline (KPBS). The first and third series were processed for PHA-L immunocytochemistry according to Gerfen and Sawchencko [18]. Briefly, sections were transferred to a solution of 2% normal rabbit serum/0.3% Triton in KPBS for 30 min and then incubated for 24 – 48 h in the primary antibody PHA-L (Vector) at a dilution of 1:2,000. After rinsing in KPBS, sections were incubated in biotinylated rabbit anti-goat IgG diluted to 1:200 in KPBS, transferred to the avidin-biotin complex immunoperoxidase (ABC Vectastain Kit; Vector), washed several times in KPBS (for 30 min), and processed for peroxidase histochemistry using a solution of 5 mg 3,3⬘-diaminobenzidine (DAB) and 0.04% H2O2 in 10 ml of KPBS. The second series of sections through the thalamus were processed for cytochrome oxidase activity according to the method described by Wong-Riley [59]. Briefly, these sections were incubated overnight at 37°C in a solution containing 4 g sucrose, 50 mg DAB, and 25 mg cytocrome C (Sigma Chemical Co., St. Louis, MO, USA; type VI) per 100 ml of PB. Sections were then rinsed in cold PB and mounted on subbed slides. The fourth series was collected and stained with thionin (0.25% in aqueous solution) for cytoarchitectonic analysis [61]. All the material was mounted on gelatin-coated slides, dehydrated, coverslipped with DPX, and examined and photographed under brightfield illumination with a Zeiss Axioscop microscope. Outlines of selected cortical sections and PHA-L-labelled axons and terminals

were drawn with the aid of a camera lucida attached to the microscope, and scanned at 300 dpi (Hewlett Packard Scan 4c) for placement in figure layouts. RESULTS A total of 37 penetrations were performed in the cortex in the present study (Fig. 1). ICMS elicited eye movements (nine penetrations), myosis (9 penetrations), whisker movements (nine penetrations) or no effect at all (eight penetrations). Only in two penetrations did ICMS elicit both whisker and eye movements. The whisker movements were obtained, for the most part, laterally to the eye movement representation in the explored cortex. The ICMS-elicited eye movements were always directed contralaterally to the stimulated hemisphere and appeared at thresholds ranging from 35 to 50 ␮A. In every penetration, at a current intensity of 50 ␮A, the ICMS evoked one movement only, except in cases R7 and R9 in which ICMS elicited both eye and whisker movements at 50 ␮A and 28 ␮A, and 50 ␮A and 32 ␮A, respectively. Injection Sites The stereotaxic coordinates of the injections in the medial frontal cortex ranged from the bregma to 1.7 mm anteriorly and from 1 mm to 1.5 mm laterally to the midline. The PHA-L injection was placed in a site of the medial frontal cortex where ICMS evoked eye movements in seven animals (injection sites within FEF; cases R1– 6 and R8), and eye and whisker movements in two cases (injection sites at the peripheral FEF; cases R7 and R9) (Fig. 1). In the reported cases, the injections were all located in the frontal cortex, area 2 (Fr2) [61]. The injection site is illustrated for two representative cases, R3 and R7, in Fig. 2. The injection sites were confined within the gray matter and contained dense deposits of brown reaction product which did not spread beyond 600 ␮m in the tangential plane. The core of the injection sites was located in layers V–VI, where ICMS had been performed. In case R5 only, the core of the injection site was located in layers III–IV. Thalamic Labelling Obtained From Injection Sites Within the FEF The labelling obtained in cases R1– 6 and R8 is summarised in Table 1. In all animals, dense patches of labelled fibres were observed in the anterior thalamic nuclei. In the representative case R3, shown in Figs. 3 and 4, the densest labelling was concentrated in the anteromedial (AM) and anteroventral (AV) thalamic nuclei.

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and the reticular thalamic nucleus. Dense labelling was found in the lateral and posterior nuclear thalamic groups, and labelled fibres were concentrated in the laterodorsal (LD), and lateroposterior (LP) nuclei, in the dorsal portion of the posterior complex (Po) (Figs. 3 and 6). A similar distribution of thalamic labelling was also observed in case R5, where the core of the injection site was located in layers III–IV (Table 1). Labelling in the Midbrain and Pons From Injections Within the FEF

FIG. 2. (A,C) Photomicrographs of two immunocytochemically processed coronal sections through frontal cortex area 2 showing the extent of the Phaseolus vulgaris leucoagglutinin iontophoretic injection in cases R3 (A) and R7 (C). (B,D) Niss1-stained sections adjacent to those shown in (A) and (C) respectively. The midline is to the right. Scale bar: 1 mm.

Less dense labelling was observed in the anterodorsal (AD) thalamic nucleus. Although the PHA-L labelling prevailed in the thalamic nuclei ipsilateral to the injected hemisphere, a weak labelling was also observed in the contralateral AM (Fig. 4A). Dense anterogradely labelled fibres were concentrated in the intralaminar thalamic nuclei. The densest labelling was located in the centromedial (CM) (Fig. 4B) and in the centrolateral (CL) nuclei of the rostral intralaminar group (Fig. 3); labelled fibres were also seen in the contralateral CL (Table 1). Labelled fibres were not observed in the paracentral nucleus (PC) of the rostral group, or in the caudal intralaminar parafascicular nucleus. In the midline thalamic nuclei, few PHA-L-labelled fibres were located in the nucleus reuniens and in the rhomboid nucleus (Fig. 3 and Table 1). Dense labelling was observed in the lateral part of the mediodorsal thalamic nucleus (MDL) of both sides (Figs. 3 and 5), and prevailed in the ipsilateral MDL. Labelled fibres were also found in the ventrolateral (VL) and ventromedial (VM) nuclei of the ventral nuclear complex ipsilateral to the injected cortex. The anterogradely labelled fibres and terminal-like elements were concentrated in the dorsal portion of VL, at the border between AV

In all animals, labelling was distributed in brainstem structures ipsilateral to the injected cortex (Table 1). Dense aggregates of PHA-L-labelled fibres were located in the anterior pretectal area (APT), and labelled fibres and terminal-like elements were distributed throughout APT. In the nucleus of Darkschewitsch (Dk) and in the area located around the third ventricle, dense labelled fibres and terminal-like elements were observed (Fig. 7). In the latter area, the labelled fibres were oriented mainly parallel to main axis of the ventricle. The distribution of the labelling in these areas was consistent in all the other cases, including case R5, in which the PHA-L injection core was located in more superficial layers (Fig. 7B). A dense aggregation of labelled fibres was observed in the superior colliculus (SC) (Table 1 and Fig. 8). Within the SC, labelled fibres and terminal-like elements were concentrated in the intermediate white matter and in the intermediate gray layer, the prevalence being lateral. In the SC, a similar distribution of labelling was observed in cases R1–R6 and R8, irrespective of the location of the injection site core (Fig. 6). Dense PHA-L labelling was detected in the central gray surrounding the aqueduct (PAG) (Fig. 9). Labelled fibres were concentrated in the dorsolateral portion of the PAG, as illustrated in Fig. 9. Some labelled fibres were also found in the pontine nuclei, laterodorsal tegmental nucleus (LDTg), in the trapezoid body nucleus, and in the parabrachial nucleus (Table 1). Light microscopic analysis of PHA-L-labelled fibres in the FEF target structures revealed the prevalence of highly varicose fibres possessing numerous collateral branches and swellings, most likely representing axon terminals and fibres lacking terminal specialisations, i.e., likely fibres-of-passage (Figs. 5, 6, 8, and 9) [52]. Thalamic Labelling Obtained From Injections of the Peripheral FEF As mentioned above, in two cases, R7 and R9, the tracer was injected in cortical sites where ICMS evoked both eye and whisker movements, and labelling was found in ipsilateral subcortical structures. As reported in Table 1, dense labelling was found in the anterior thalamic nuclei. Labelled fibres were concentrated in AD and AV (Fig. 3). Dense labelled terminal like-elements were also concentrated in the ventral and central portions of VL and in VM (Fig. 3). Furthermore, the anterograde labelling was located in the lateral and posterior nuclear thalamic groups, where labelled fibres were concentrated in the medial portion of LD and LP, and in Po (Figs. 3 and 10). A few PHA-L-labelled fibres were also observed at the midline within Re and in the intralaminar CL. No labelled fibres were observed in MDL. Labelling in the Midbrain and Pons From Injections of the Peripheral FEF Labelled fibres were located in the posterior hypothalamic nucleus; aggregates of PHA-L-labelled fibres were located be-

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GUANDALINI TABLE 1 DISTRIBUTION AND SEMI-QUANTITATIVE ANALYSIS OF DENSITY OF THE TERMINAL LABELLING IN THALAMUS AND BRAINSTEM STRUCTURES AFTER PHASEOLUS VULGARIS LEUCOAGGLUTININ (PHA-L) INJECTIONS Efferent Structures

Thalamus Anterodorsal nucleus Anteromedial nucleus Anteroventral nucleus Central medial nucleus Centrolateral nucleus Gelatinosus nucleus Lateral posterior nucleus Laterodorsal nucleus Mediodorsal nucleus Lateral mediodorsal nucleus Medial mediodorsal nucleus Paracentral nucleus Parafascicular nucleus Paratenial nucleus Paraventricular nucleus Anterior paraventricular nucleus Posterior paraventricular nucleus Posterior thalamic nuclear group Reticular nucleus Reuniens nucleus Rhomboid nucleus Ventrolateral nucleus Ventromedial nucleus Ventroposterior nucleus Lateral ventroposterior nucleus Medial ventroposterior nucleus Hypothalamic nucleus Brainstem Anterior pretectal area Dorsal anterior pretectal area Interstitial nucleus of Cajal Laterodorsal tegmental nucleus Nucleus of Darkschewitsch Nucleus of Edinger-Westphal Superior colliculus Intermediate gray layer Intermediate white layer Optic nerve layer Parabracheal nucleus Periaqueductal gray Pontine nuclei Red nucleus Rubrospinal tract Superior cerebellar nucleus Trapezoid body nucleus

Case R1

Case R2

Case R3

Case R4

Case R5

Case R6

Case R7

Case R8

Case R9

⫹⫹ ⫹⫹⫹ c⫹ ⫹⫹⫹ ⫹⫹ ⫹⫹ c⫹ – ⫹⫹ ⫹⫹

⫹⫹ ⫹⫹ c⫹ ⫹⫹ ⫹⫹⫹ ⫹⫹⫹ c⫹ – ⫹⫹ ⫹⫹

⫹⫹⫹ ⫹⫹⫹ c⫹ ⫹⫹ ⫹⫹⫹ ⫹⫹⫹ c⫹ – ⫹⫹⫹ ⫹⫹

⫹⫹⫹ ⫹⫹ c⫹ ⫹⫹ ⫹⫹ ⫹⫹ c⫹ – ⫹⫹ ⫹⫹

⫹⫹ ⫹⫹ c⫹ ⫹⫹⫹ ⫹⫹⫹ ⫹⫹⫹ c⫹ – ⫹⫹⫹ ⫹⫹

⫹⫹⫹ ⫹⫹⫹ c⫹ ⫹⫹ ⫹⫹ ⫹⫹ c⫹ – ⫹⫹ ⫹⫹

⫹ – ⫹

⫹ – ⫹

⫹ – ⫹⫹ ⫹⫹

⫹⫹ ⫹⫹ c⫹ ⫹⫹ ⫹⫹ ⫹⫹⫹ c⫹ – ⫹⫹ ⫹⫹

⫹ – ⫹⫹ ⫹⫹

⫹⫹⫹ c⫹ – – – –

⫹⫹ c⫹ – – – –

⫹⫹⫹ c⫹ – – – –

⫹⫹ c⫹ – – – –

⫹⫹ c⫹ – – – –

⫹⫹ c⫹ – – – –

– – – – –

⫹⫹ c⫹ – – – –

– – – – –

– – ⫹⫹ – ⫹ ⫹ ⫹ ⫹

– – ⫹⫹⫹ – ⫹ ⫹ ⫹ ⫹

– – ⫹⫹⫹ – ⫹ ⫹ ⫹ ⫹

– – ⫹⫹ – ⫹ ⫹ ⫹ ⫹

– – ⫹⫹ – ⫹ ⫹ ⫹ ⫹

– – ⫹⫹ – ⫹ ⫹ ⫹ ⫹

– – ⫹⫹ – ⫹ – ⫹⫹⫹ ⫹

– – ⫹⫹ – ⫹ ⫹ ⫹ ⫹

– – ⫹⫹ – ⫹ – ⫹⫹⫹ ⫹

– – –

– – –

– – –

– – –

– – –

– – –

– – ⫹⫹

– – –

– – ⫹⫹

⫹⫹ ⫹⫹ ⫹ ⫹ ⫹ ⫹

⫹⫹ ⫹⫹ ⫹ ⫹ ⫹⫹ ⫹

⫹⫹ ⫹⫹ ⫹ ⫹ ⫹⫹ ⫹

⫹⫹ ⫹⫹ ⫹ ⫹ ⫹ ⫹

⫹⫹ ⫹⫹ ⫹ – ⫹⫹ ⫹

⫹⫹ ⫹⫹ ⫹ – ⫹ ⫹

⫹ – – ⫹ ⫹⫹⫹ ⫹

⫹⫹ ⫹⫹ ⫹ ⫹ ⫹⫹ ⫹

⫹ – – ⫹ ⫹⫹⫹ –

⫹⫹⫹ ⫹ – – ⫹⫹ ⫹ – – – ⫹

⫹⫹ ⫹ – ⫹ ⫹⫹ ⫹ – – – –

⫹⫹⫹ ⫹ – ⫹ ⫹⫹ ⫹ – – – ⫹

⫹⫹ ⫹ – – ⫹⫹ ⫹ – – – ⫹

⫹⫹ ⫹ – – ⫹⫹ – – – – ⫹

⫹⫹⫹ ⫹ – – ⫹⫹ – – – – –

⫹⫹ ⫹⫹⫹ ⫹

⫹⫹ ⫹ – ⫹ ⫹⫹ – – – – –

⫹⫹ ⫹⫹⫹ ⫹

⫹⫹ ⫹⫹ ⫹⫹ ⫹ ⫹

⫹ ⫹⫹ ⫹⫹ ⫹ ⫹

–, no evident PHA-L-labelling; ⫹, 1–250 PHA-L-labelled puncta per section; ⫹⫹, 251–1,000; ⫹⫹⫹, more than 1,000; c, contralateral.

tween 3.8 mm and 4.3 mm anteriorly to the bregma, between the third ventricle and the mammillothalamic tract according to Paxinos and Watson [41]. Some labelled fibres and terminallike elements were distributed in the centromedial portion of the APT. Dense labelling was located in the Dk nucleus (Fig. 11), which contained labelled fibres and terminal-like elements throughout its extent. Some labelled fibres were located in the accessory oculomotor nucleus, in the red nucleus and in the

superior cerebellar peduncle. The densest labelling was found within SC (Fig. 12). PHA-L-labelled fibres and terminal-like elements were concentrated in the intermediate white matter and the lateral portion of the intermediate gray layer throughout the thickness of the SC. Dense labelled fibres were also detected in the PAG and in the pontine nuclei. Less dense labelling was found in the LDTg. Light microscopic analysis of PHA-L-labelled fibres in structures targeted by efferents of the peripheral FEF revealed a pre-

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FIG. 3. Camera lucida drawing of the distribution of anterograde thalamic labelling (dots) after a iontophoretic injection of Phaseolus vulgaris leucoagglutinin in two representative cases. Abbreviations: AD, anterodorsal thalamic nucleus; AM, anteromedial thalamic nucleus; APT, anterior pretectal area; APTD dorsal part; AV, anteroventral thalamic nucleus; CG, central (periaqueductal) gray; CL, centrolateral thalamic nucleus; CM, central medial thalamic nucleus; Dk, nucleus of Darkschewitsch; EW, accessory oculmotor nucleus (Edinger-Westphal); fr, fasciculus retroflexus; G, nucleus gelatinosus of the thalamus; InC, interstitial nucleus of Cajal; InG, intermediate gray layer of the superior colliculus; InWh, intermediate white layer of the superior colliculus; LD, laterodorsal thalamic nucleus; LDTg, laterodorsal tegmental nucleus; LHb, lateral habenular area; LP, lateral posterior thalamic nucleus; MD, mediodorsal thalamic nucleus; MDL, mediodorsal thalamic nucleus, lateral part; ml, medial lemnicus; Op, optic nerve layer of the superior colliculus; PC, paracentral thalamic nucleus; PF, parafascicular thalamic nucleus; PH, posterior hypothalamic nucleus; Pn, pontine nuclei; Po, posterior thalamic nuclear group; PT, paratenial thalamic nucleus; PVA, paraventricular thalamic nucleus, anterior part; PVP, paraventricular thalamic nucleus, posterior part; py, pyramidal tract; R, red nucleus; Re, thalamic nucleus reuniens; Rh, rhomboid thalamic nucleus; rs, rubrospinal tract; Rt, reticular thalamic nucleus; SC, superior colliculus; scp, superior cerebellar peduncle (brachium conjunctivum); sm, stria medullaris; VL, ventrolateral thalamic nucleus; VM, ventromedial thalamic nucleus; VPL, ventroposterior thalamic nucleus, lateral part; VPM, ventroposterior thalamic nucleus, medial part.

vailing type of highly varicose fibres possessing numerous collateral branches and swellings, which most likely represented axon terminals as well as fibres-of-passage.

DISCUSSION The medial frontal cortex has been widely investigated in the rat [10,42– 44,56], and has appeared cytoarchitectonically and

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GUANDALINI connectionally heterogeneous. The present tract-tracing experiments show that in the rat FEF, a discrete area of the medial frontal cortex cytoarchitectonically identified as Fr2 where ICMS causes eye movements, sends projections to several structures of the thalamus, midbrain, and pons. Cortical sites within Fr2, located at the lateral border of FEF, i.e., the peripheral FEF where ICMS elicits eye and whisker movements, also send projections to several thalamic, midbrain and pontine structures although the projection pattern differs from that seen for FEF. Thalamic Connections

FIG. 4. Bright-field (A) and dark-field (B) photomicrographs of anterogradely preterminal and terminal labelling in the anterior thalamic nuclei (A) and intralaminar thalamic nuclei (B), respectively, after Phaseolus vulgaris leucoagglutinin injection in case R3. Scale bar: 200 ␮m. Abbreviations: AD, anterodorsal thalamic nucleus; AM, anteromedial thalamic nucleus; AV, anteroventral thalamic nucleus; CM, central medial thalamic nucleus; PT, paratenial thalamic nucleus; SM, stria medullaris.

The main FEF terminal fields are concentrated in AD, AM, AV,CM, CL, MDL, LD, LP, and Po. Several previous investigators have studied the thalamic connections of the medial frontal cortex in the rat [10,43– 45]. Reep et al. [43,45] found that the thalamic connections of the subdivision referred to as the medial agranular cortex (AGm) [16] or Fr2 [61] are organised in a continuous gradient whereby more caudal portions of AGm have connections with more lateral and caudal regions of the thalamus [43]. Many of the thalamic projections observed in the present report are consistent with those reported by Reep and Corwin [43] and correspond to those originating in the rostral portion of the caudal AGm area. In the present report, FEF was found to be located in Fr2 between bregma and 1.7 mm anteriorly. Connections to the anterior thalamic nuclei observed in the present report were not documented by Reep and Corwin [43] but are consistent with the findings previously reported by Hicks and Huerta [27] who investigated the connectivity of the rostral and caudal parts of Fr2, ICMS eliciting body movements in the former and eye and eyelid movements in the latter [16,40,49]. However, the AM, AV, LD, and LP thalamic nuclei were found to project to the caudal Fr2 [27]. The anterior thalamic nuclei also project to the cingulate cortex, which is involved in controlling emotion [13,17]. More recently Zeng and Stuesse [60] proposed that two cytoarchitec-

FIG. 5. Photomicrographs illustrating the projections of the frontal eye field site (case R3) to the lateral portion of the mediodorsal thalamic nucleus (MDL). (A) Coronal section illustrating the location of anterogradely labelled fibres in MDL. (B) Higher magnification of (A) illustrating corticothalamic labelled fibres which exhibit numerous swellings along their course, interpreted as boutons en passant (arrow) and stalked boutons (arrowheads). Scale bars: 200 ␮m in (A) and 10 ␮m in (B). Abbreviations: CL, centrolateral thalamic nucleus; MD, mediodorsal thalamic nucleus; MDL, mediodorsal thalamic nucleus.

EYE FIELD CONNECTIONS IN THE RAT MEDIAL FRONTAL CORTEX

FIG. 6. Photomicrographs illustrating the projections of the frontal eye field site (case R3) to the posterior nuclear thalamic groups.(A) Coronal section illustrating the location of the anterogradely labelled fibres in the lateroposterior nucleus (LP) and in the dorsal portion of the posterior nucleus complex (Po). (B) Higher magnification illustrating the dense field of labelled preterminal elements, the distinct axonal morphology of which is illustrated at a higher magnification in C showing boutons en passant (arrows) and stalked boutons (arrowheads). Stars in (A) and (B) indicate the same blood vessel as reference point. Scale bars: 500 ␮m in (A), 100 ␮m in (B), and 10 ␮m in (C). Abbreviations: LH, lateral hypothalamic area; LP, lateral posterior thalamic nucleus; Po, posterior thalamic nuclear group.

tonic subdivisions of the cingulate cortex, Cg3 and Cg1/Cg2 as described by Zilles [61], represent visceral (Cg3) and somatic (Cg1/Cg2) motor areas, respectively. The cingulate cortex, compared with AGm, proves to have fewer thalamic connections with the VL and many more connections with the anterior thalamic nuclei [46]. Overlapping connections between AGm and cingulate cortex are documented in the SC [10,37,45,46]. In the rat the rostral intralaminar group composed of the CM, PC, and CL nuclei, receives cortical afferents from a variety of areas, including the parietal, cingulate and retrosplenial cortical

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areas and FEF [4,5,32,42]. In the monkey, the intralaminar nuclei play a role in inducing gaze, commanding visual orientation, and controlling eye movements [50,51]. In the rat, the present finding of FEF connections with CL and CM nuclei support the role of the intralaminar nuclei in controlling eye movements. However, FEF projections to PC were not identified anterogradely in the present study, even though a large number of retrogradely labelled neurones were described in PC after horseradish peroxidase injections in the rat frontal pole and parietal cortex [27,43]. On the other hand, functionally the frontal cortex is not a homogeneous structure [10,27] and, within the frontal cortex, FEF exhibits a distinct pattern of connections [23,27,46]. In the monkey, PC has instead relatively scanty connections with FEF [29], whereas the CL and CM intralaminar nuclei receive projections from FEF [55]. The present study showed that FEF projections terminate primarily in MDL, which proves to be a thalamic target of AGm projections. In the monkey, MD is composed of three cytoarchitectonically different segments, the medial, lateral, and paralamellar portions, which establish reciprocal connections with orbitofrontal areas, the dorsolateral prefrontal cortex and FEF, respectively [29]. In the rat, there are no clear cytoarchitectonic criteria defining MD subdivisions, although separate segments of this nucleus are reciprocally connected with different subregions of the frontal cortex [21]. In the present study, two MD segments have been identified on the basis of their connectivity, i.e., a medial and a lateral segment, the latter receiving dense projections from FEF. Supposing that the lateral segment of the rat corresponds to that of the monkey, the corticothalamic projections from FEF in the rat could be similar to those of the monkey. On the other hand, the lateral part of the monkey MD is implicated in visuomotor function [30,50,51]. The present experiments show that the cortical sites of the peripheral FEF send projections to several areas of the thalamus and in particular, to the anterior, posterior, and ventral thalamic nuclei. The first two nuclear groups also received projections from the FEF, although less dense and with a different topographical arrangement. The VL and VM nuclei were here found to be targets of the peripheral FEF projections. Reep and Corwin [43] found a uniform distribution of the VL projections to the AGm area, and Hicks and Huerta [27] reported that the VL projects to the rostral, but not the caudal, Fr2. However, the AGm area is not the sole target of VL, because thalamic connections with VL are widely documented in the lateral agranular field, AG1 [15]. In the rat, AG1, corresponding to Fr1 [61], is connected with VL, VM, PM, and CL [16], while the thalamic projections from the barrel cortex in the somatosensory cortex have targets in the reticular, ventrobasal, and posterior thalamic nuclei [28]. Moreover, the present data indicate that the peripheral FEF projects sparsely to the intralaminar thalamic nuclei and does not project to the MDL. The MDL is reciprocally connected with the AGm [27,43] and does not appear to have connections with AG1 [1,14,33]. Therefore, the peripheral FEF projections could be similar to those that arise from the AG1 area. On the other hand, the cortical sites where ICMS elicits both eye and whisker movements were located in Fr2, and not in Fr1. One explanation for the incompatibility of the present findings with previously reported cytoarchitectonic subdivisions is that the boundary between functionally different parts of the cortical areas varies and is subject to overlapping [49]. Connections to Midbrain and Pons Several investigators have studied the connectivity of the frontal cortical areas with midbrain and pons structures, and many of these structures are involved in visual functions [35–37,47,56]. Our results show that, in the midbrain and pons, the APT area, Dk

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FIG. 7. (A,B) Camera lucida drawing of the core of the cortical Phaseolus vulgaris leucoagglutinin (PHA-L) injection site of case R4 (A) and case R5 (B) and anterogradely labelled fibres in the midbrain nucleus of Darkschewitsch (Dk) in the same cases. (C) Camera lucida reconstruction of one PHA-L-labelled axon, from the Dk in case R3, showing an axonal collateralisation and numerous boutons en passant. The levels of the sections in respect to the bregma are indicated.

nucleus, SC, and PAG are the main targets of projections arising both from FEF and peripheral FEF. Stuesse and Newman [56] reported that, in the rat, the AGm projects to several structures, including the anterior APT, interstitial nucleus of Cajal, nucleus of Dk, intermediate and deep superior collicular layers. Moreover, differences were observed in connections between the rostral and caudal portions of the AGm. Leichnetz and Gonzalo-Ruiz [36] and Leichnetz and Goldberg [35] found that the cortical region of the rat dorsomedial frontal shoulder cortex (medial precentral/anterior cingulate cortices of Krettek and Price [33]) contains lamina V pyramidal neurones whose axons collateralise, projecting to the oculomotor complex, SC, and medial pontine reticular formation. This pattern of projections has been observed in the monkey as well [34]. In the present study, an injection site core located in the superficial layers, did not result in different labelling from the other FEF injections. In the present report, dense projections to APT have been observed, especially those arising from the FEF cortical sites where ICMS elicited eye movements, as well as to in the Dk nucleus, especially from the lateral border FEF cortical sites where ICMS elicited both eye and whisker movements. The functional role of APT has been investigated in the monkey; in this species APT receives direct projections from the retina, and has direct connections with the oculomotor complex, contributing to the control of pupillary light reflex [8,34]. The Dk nucleus is partly embedded in the PAG and, together with the nucleus of the posterior commissure and the interstitial Cajal nucleus, constitutes the accessory oculomotor nuclei or oculomotor complex. The peripheral FEF cortical sites were also found to project to the nucleus of Edinger-Westphal and red nucleus, superior cerebellar nucleus, and pontine nuclei. The same patterns of distribution were observed in a previous study upon PHA-L tracer injections into the whisker area representation of the rat motor cortex [39]. Previous investigations have documented the connections link-

ing SC and frontal cortical areas [2,25,26,40,45], and, in particular, the projections of the prelimbic, anterior cingulate, and somatic cortices to the intermediate and deep layers of the SC. The medial three-quarters of the intermediate gray layer of the SC were found to receive projections arising from prelimbic and anterior cingulate cortices [2]. The AGm terminal field was found to be localised to the lateral one-quarter of the SC intermediate gray layer [45]. In the SC, the superficial layer receives direct fibre connections from the retina in a retinotopic manner, while the intermediate and deep layers emit descending fibres to the lower brainstem. The present findings show that the FEF and the peripheral FEF cortical sites send the densest contingent of projections to the SC. The terminal fields prevailed in the lateral intermediate gray and white layers of the SC. In particular, the intermediate white layer was recipient of the projections arising from the peripheral FEF cortical sites, in agreement with previous data [45]. The termination pattern within the SC was similar to that previously reported in the macaque monkey [29,55]. The PAG is a functionally heterogeneous region which plays a role in the regulation of complex sensory, motor, and autonomic functions and many brain regions are reciprocally connected with the PAG [3]. The PAG consists of four anatomical divisions: medial, ventrolateral, dorsolateral, and dorsal [3,11]. In the present study, the dorsolateral division was found to be the recipient of the FEF projections. It has been shown that this division preferentially projects to the CL, the paraventricular thalamic nuclei, and the anterior hypothalamic area [9]. On the other hand, the CL thalamic nucleus was also found to be a target of FEF projections. The Dk nucleus, the SC, and PAG are connected with the medial cerebellar nucleus and are, therefore, involved in controlling eye movements [20]. In primates, all these structures are involved in the mechanisms controlling a variety of eye movements [55]. In the present report, the core of the injection sites were

EYE FIELD CONNECTIONS IN THE RAT MEDIAL FRONTAL CORTEX

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FIG. 8. Camera lucida drawing of anterogradely labelled fibres in the superior colliculus (SC) after Phaseolus vulgaris leucoagglutinin (PHA-L) injections within frontal eye field in three different cases. (A) Case R8 (C) case R3, and (D) case R6, illustrate the camera lucida drawings of coronal sections through SC, the framed region shown at higher magnification, and the injection site core. The injection site core of case R3 in C is shown in Fig. 1. (B) Photomicrograph of anterogradely labelled fibres in the SC after the PHA-L injection in case R8, showing the dense distribution of the labelled puncta. Scale bar in (B): 20 ␮m. Abbreviations: InG, intermediate gray layer of the superior colliculus; InWh, intermediate white layer of the superior colliculus; Op, optic nerve layer of the superior colliculus.

located in the deep cortical layers, except for case R5, where the core of the injection site was found to be located in the superficial layers. The vast majority of the axons projecting to the thalamus, brainstem, and pons arise from the deep layers of the medial frontal cortex [12,52]. Yet in the present report the projection patterns of case R5 were similar to those of the other cases examined. It may be that uptake of the tracer deposited by a small number of neurones in the deep layers was sufficient to give a labelling pattern similar to that obtained when a much larger number of deep layer neurones was involved.

Functional Considerations In the present report, two different patterns of efferent connections were found: one belonging to the FEF, the other to the peripheral FEF. This is in agreement with a previous study [24], and functional microstimulation is confirmed as the only means to identify the FEF sites. Unlike the corticocortical [24] and corticothalamic projections of the FEF sites, the FEF and the peripheral FEF projections to the brainstem share common targets in the Dk nucleus, the SC, and the PAG and there is no difference in the topographical arrangement of the terminal fields within the SC

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FIG. 9. (A) Camera lucida drawing of anterogradely labelled fibres found in the dorsolateral portion of the periaqueductal gray after the Phaseolus vulgaris leucoagglutinin injection in the frontal eye field. (B) Photomicrograph from the area framed in (A) illustrating the anterogradely labelled fibres in the dorsolateral portion of the periaqueductal gray. Scale bar: 20 ␮m. Abbreviations: Aq, cerebral aqueduct; GC, central (periaqueductal) gray; CGD, central grey, dorsal part; DR, dorsal raphe nucleus.

structures. In the rat, the whisker constitutes a major tactile sense organ for spatial orientation, exploration, and target location. The intermediate and deep layers of the SC represent one of the targets of the afferent projections from the receptor apparatus to the cerebral cortex. On the other hand, the SC is involved in the control of eye movements: electrical stimulation of the SC in rodents, carnivores, and primates elicits saccadic eye movements [38,47,48] and stimulation of the SC evokes not only eye movements but also whisker movement [38]. The SC is closely associated with the organisation of visual and body-orienting movements [54,58] and mechanisms of attention, arousal, and postural orienting [22,42] because, throughout the various collicular layers, visual afferents are linked with afferents related to other sensory modalities as well as with non-sensory afferents. These inputs are

GUANDALINI

FIG. 10. Photomicrographs of the lateral and posterior nuclear thalamic groups after the Phaseolus vulgaris leucoagglutinin injection at the peripheral FEF in case R7. (A) Coronal section stained for cytochrome oxidase. (B) Section adjacent to (A), showing anterogradely labelled fibres in the medial portion of lateroposterior nucleus (LP), illustrating in (C) dense labelled puncta (arrows). Scale bars: 1 mm in (A), 50 ␮m (B), and 10 ␮m in (C). Abbreviations: CL, central thalamic nucleus; LH, lateral hypothalamic area; LP, lateral posterior thalamic nucleus; Po, posterior thalamic nuclear group; VPM, ventrosposterior thalamic nucleus, medial part.

orchestrated in a topographic fashion and lead to premotor neurons, key elements in the generation of saccadic eye movements and orientation movements in general [31]. The Dk, SC, and PAG are interconnected structures [3] constituting the common recipient of the projections from the FEF and lateral border FEF sites. As documented ill the present study, in the rat these nuclei constitute the anatomical substrate where the peripheral sensory information perceived from the whiskers is integrated through intrinsic connections and generates the eye movements during exploration and orienting behaviour.

EYE FIELD CONNECTIONS IN THE RAT MEDIAL FRONTAL CORTEX

FIG. 11. Camera lucida drawing of the anterogradely labelled fibres in the nucleus of Darkschewitsch (Dk) after the Phaseolus vulgaris leucoagglutinin injection of the peripheral frontal eye field in case R9.

In conclusion, the present study has elucidated the organisation of the brainstem efferent connections of the rat FEF. The brainstem efferent connections of the FEF seem to share a similar organisation in the rat and in the monkey. Yet common recipients in the brainstem for whiskers and eye movements confirm that in the rat the saccadic eye movements are a means for exploring the surrounding environment and a useful tool in orienting behaviours, rather than quickly directing foveas to a target of interest in visual space, as occurs in primates. Despite the similarity of the anatomical connections established between the rat and monkey FEF and other cerebral structures, the contribution of the cortical area responsible for controlling eye movements differs in the rat, suggesting greater involvement of the subcortical structures. ACKNOWLEDGMENTS

This work was supported by the Italian Ministero dell’Universita` e della Ricerca Scientifica e Tecnologica (MURST, Fund 40% and 60%). The author is grateful to Prof P. Barbaresi for valuable advice and technical assistance, and Mr. V. Muzzioli for his assistance with the preparation of the figures.

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