Brain Research, 580 (1992) 137-146 (~) 1992 Elsevier Science Publishers B.V. All rights reserved. 0006-8993/92/$05.00
137
BRES 17716
Electrophysiological and anatomical studies on thalamic mediodorsal nucleus projections onto the prefrontal cortex in the cat Ikuo Tanibuchi Department of Integrative Brain Science, Faculty of Medicine, Kyoto University, Kyoto (Japan) (Accepted 24 December 1991) Key words: Thalamocortical projection; Mediodorsal nucleus; Prefrontal cortex; Field potential; Horseradish peroxidase
Electrical stimulation of the mediodorsal nucleus (MD) of the thalamus elicited field potentials in the gyrus proreus (PRO), frontalis (FR), rectus (RE) and cinguli anterior (CIant) of the ipsilateral prefrontal and adjacent cortical areas in cats. The results of a laminar field potential analysis indicate that the field potentials can be regarded as a combination of deep and superficial thalamocortical responses. By injecting horseradish peroxidase (HRP) into the MD, HRP-labeled terminals were distributed in the prefrontal and adjacent cortical areas where the field potentials were elicited. Densely labeled terminals in cortical layer I were distributed where the superficial thalamocortical responses were prominent, while those in layers III-V were distributed in the areas where the deep thalamocortical responses were prominent. INTRODUCTION It is generally thought that the mediodorsal nucleus (MD) of the thalamus, in connection with the prefrontal cortex, plays an important role in learning, memory and olfactionl,13, 24,34-36. The frontal cortical areas where MD neurons project were defined as the prefrontal cortex by Rose and Woolsey 23 ('orbitofrontal cortex' in their terminology). Anatomical studies in the rat, cat and monkey, however, disclosed that the MD also projects to cortical areas other than the prefrontal cortex 4'6'9'1°'17'18'2°'21. Because of the disagreement among the morphological studies on the MD-prefrontal projections 14'15'19, the precise boundary of the prefrontal cortex has not yet been established in the cat. There are also some differences among these morphological findings in the topography of the thalamocortical projections from the subdivisions of the MD to the prefrontal cortex. The first aim of the present study was to determine the MD projection areas in the prefrontal and adjacent cortical areas of cats with both electrophysiological and morphological methods, i.e., an analysis of cortical field potentials elicited by MD stimulation, and anterograde axonal transport study after horseradish peroxidase (HRP) injection into the MD. Thalamocortically evoked potentials in the motor and sensory cortices, as well as in the parietal association
cortex have been investigated in cats 11'12'16'25-30'32. According to an analysis of laminar field potentials, thalamocortically evoked potentials in these cortical areas were found to consist of two kinds of elementary responses, superficial and deep thalamocortical potentials 3°. Superficial thalamocortical responses are surface negative-depth positive (sN-dP) potentials, due mainly to excitatory postsynaptic currents (EPSCs), generated by thalamocortical afferent fibers activating superficial parts of the apical dendrites of pyramidal neurons. The surface negative potentials usually turn positive at a depth of 0.2 mm. Thalamocortical afferents reaching layer I directly are considered to be the morphological substrates of the superficial thalamocortical projections 7' 8,31. Deep thalamocortical responses are surface positive-depth negative (sP-dN) potentials, which usually turn negative at a depth of 0.25 mm. They can be accounted for by the EPSCs, which are generated by thalamic afferents making synapses on deep parts of the apical dendrites and the somata of pyramidal neurons. Thalamocortical afferents terminating in layers I I I - V of the cerebral cortex may correspond to these deep thalamocortical projections 7'8'26'30'31. Thalamocortical responses in the prefrontal and adjacent cortices evoked by MD stimulation have not yet been studied in terms of the superficial and deep thalamocortical responses. Thus, the second aim of the present study was to examine whether the two elementary responses, the superfi-
Correspondence: I. Tanibuchi, Department of Integrative Brain Science, Faculty of Medicine, Kyoto University, Yoshidakonoe-cho, Sakyoku, 606, Kyoto, Japan.
138 cial and deep responses, can be attributed to the thalamocortical projection fibers which terminate in layer I and layers III-V, respectively, of the prefrontal and adjacent cortices.
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MATERIALS AND METHODS
Electrophysiology Thirty-six adult cats of both sexes, weighing 2.5-5.2 kg, were anesthetized by sodium pentobarbital (30 mg/kg, i.v.) and fixed to a stereotaxic instrument. A craniotomy was performed to expose the lateral or mesial surface of the frontal cortex. These lateral and mesial surfaces were examined in separate experiments. For the experiments examining the mesial surface, the frontal cortex in one side was aspirated out to expose the mesial surface of the contralateral prefrontal cortex. Six to nine concentric, stimulating electrodes (o.d. 0.5 ram, the central core extruding by about 0.5 ram) were introduced vertically into the MD ipsilateral to the exposed prefrontal cortex. The MD was then stimulated at 1 Hz, with a current pulse of 300/~s duration and less than 120/~A. The stimulation sites were examined histologically after every experiment. A glass microelectrode of 2-4 Mf~ DC-resistance filled with 0.75 M sodium acetate (CH3COONa) was inserted into the prefrontal and adjacent cortices perpendicularly to their surface, to record the field potentials. This microelectrode was advanced step by step to a depth of 2.5 mm while field potentials were recorded at various depths. The exposed surface of the cortex was constantly bathed in warm Ringer's solution to protect the cortex from cooling and drying. The body temperature was maintained between 36 and 37°C throughout the surgical and recording procedures.
Anatomy Thirty-two adult cats of both sexes, weighing 2.6-5.1 kg, were used for the HRP study. Under sodium pentobarbital anesthesia (30 mg/kg, i.v.), craniotomies were done in the cats. Wheat germ agglutinin--horseradish peroxidase (WGA-HRP) was injected into the unilateral MD in order to label the terminals of the thalamocortical projection fibers in the ipsilateral prefrontal and adjacent cortices by anterograde axonal transport. The injections of 5% W G A - H R P dissolved in 0.9% saline were made stereotaxically, by using a 1 /~1 Hamilton microsyringe; the volume varied between 0.02 and 0.06/~1 per injection. Following the survival time of 28-48 h, the animals were deeply anesthetized with sodium pentobarbital. They were then perfused intracardially with 500-750 ml of 7% formalin in 0.1 M phosphate buffer (pH 7.6), followed by 0.1 M phosphate buffer containing 10% sucrose. The brains were removed immediately after the perfusion and soaked in 0.1 M phosphate buffer containing 30% sucrose at 4°C for 3-5 days. The frozen brains were then cut serially into frontal sections of 70/~m thickness through the thalamic injection sites and the prefrontal and adjacent cortices. Sections of the thalamus were treated with 3,3'-diaminobenzidine-tetrahydrochloride (DAB) according to the method of Streit and Reubi 33. They were counterstained with cresyl violet, and then examined microscopically under bright-field illumination. The injection site was defined as the region in which dense HRP reaction product was visible under bright-field illumination. Thalamic injection sites were reconstructed using an overhead projector. The sections from the prefrontal and adjacent cortices were treated with benzidine dihydrochloride (BDHC) according to the method of DeOlmos and Heimer 3. They were examined microscopically under dark-field illumination without counterstaining in order to avoid fading of the labeled terminals, and were then counterstained with neutral red in order to reconstruct the laminar structure of the cortex. The distribution of the labeled terminals was charted on the drawings taken from the counterstained sections. Nomenclature and delineation of the thalamic nuclei followed the atlas by Berman and Jones2.
B
Fig. I. Diagrams showing the subdivisions of the prefrontal and adjacent cortices (PRO, PRSm, FR, RE and CIant), based on the atlas by Reinoso-Suarez22. A: the lateral and mesial views of the cat cortex. B: the frontal sections corresponding to the six arrows in each view in A. The same frames are used in Figs. 5-7B.
The subdivisions of the prefrontal and adjacent cortices, used in the following sections, are shown in Fig. 1.
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Fig. 2. Examples of laminar field potentials recorded at various depths (0.0-2.5 mm) from the surface at a, b and c, as indicated in Fig. 3. About 6 sweeps were superimposed on each trace. The depth for column b is the same as for a. a: deep thalamocortical response, b: a succession of deep and superficial thalamoeortical responses, c: superficial thalamocortical response. Voltage calibration for columns a and b is 1 mV, and is 0.5 mV for column c. The time scale for all records is 50 ms. Depths are indicated in millimeters on the left of a and c.
139 threshold deep+superficial •
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Fig. 3. A-1 and A-2 show the distribution of the field potentials which MD stimulation elicited in the mesial and lateral regions of the prefrontal and adjacent cortices, respectively. Open circles, filled squares and filled circles show deep, superficial and a succession of deep and superficial thalamocortical responses, respectively. Their sizes are inversely proportional to the thresholds of the field potentials elicited, i.e, large marks show field potentials with low thresholds. Crosses show no response. B-1 and B-2 show the stimulation sites in the MD which evoked field potentials in the mesial and lateral regions of the prefrontal and adjacent cortices, respectively. The frontal sections of the thalamus through the MD (+10.2, 9.5, 9.1, 8.3, 7.9) were ipsilateral to the prefrontal cortex where the field potentials were recorded. The number above each coronal section (right side; medial) indicates the distance from the interaural plane. Arrows accompanied by asterisks indicate the stimulation sites in the MD (MDrvl) which evoked the field potentials with the lowest threshold.
140 RESULTS
Electrophysiology Fig. 2 shows examples of field potentials recorded from microelectrode tracks at sites a, b and c in the prefrontal and adjacent cortices (see Fig. 3A-l, A-2). In column a, a single stimulation of the MD produced surface positive (0.00-0.25 mm) and depth negative (0.252.50 mm) (sP-dN) potentials. The reversal of polarity was at a depth of about 0.25 mm. The latency was about 2.5 ms. These characteristics coincide with those of the deep thalamocortical response. The field potentials in column c have the same characteristics as the superficial thalamocortical response. The reversal level was at a depth of about 0.2 mm, and the latency was about 17.8 ms. The evoked potentials in column b can be interpreted as a succession of deep and superficial thalamocortical responses. The superficial thalamocortical response is slightly larger than the deep thalamocortical response. The reversal level of the deep thalamocortical response was at a depth of about 0.25 mm, whereas that of the superficial thalamocortical response apppears to be slightly more superficial than 0.25 mm. The latencies of the deep and superficial thalamocortical responses were about 9.3 ms and about 22.0 ms, respectively. Fig. 3 A-1 and A-2 show the distribution of the field potentials elicited in the prefrontal and adjacent cortical areas by MD stimulation with a current of less than 120 /~A. Field potentials were elicited in the gyrus frontalis (FR) (including area 6), gyrus rectus (RE), gyrus cinguli anterior (Clant) (paralimbic region) and the gyrus proreus (PRO). In most electrode tracks, the initial positive potentials followed by the negative ones were recorded at the surface. The surface positive potentials turned negative at a depth of about 0.25 mm, while the surface negative potentials turned positive at a depth of about 0.20 mm. Most potentials evoked by MD stimulation were therefore regarded as deep thalamocortical responses followed by superficial thalamocortical responses. Pure deep thalamocortical responses were elicited in the rostral part of the RE (REr), and the dorsolateral and ventral parts of the PRO (PROdl and PROv). Pure superficial thalamocortical responses were elicited in the caudodorsal part of the FR (FRcd), and the Clant. The latencies of the deep and superficial thalamocortical responses were 1-16 ms (mean: 6.3 ms), and 14-25 ms (mean: 18.8 ms), respectively. The field potentials in the mesial prefrontal and adjacent cortices (FR, RE) were evoked by stimulation of the whole rostrocaudal extent of the MD (Fig. 3B-l), while +hose in the lateral prefrontal cortex (PRO) were evoked by stimulation of the rostral part of the MD (Fig. 3B-2). The threshold for eliciting field potentials tended to be low
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Fig. 4. Distribution of the deep thalamocortical (filled circles in A) and superficial thalamocortical (filled squares in B) responses in the prefrontal and adjacent cortical areas of the cat, evoked by MD stimulation with a current of 120 ~A. Their relative sizes are in proportion to the amplitude of the field potentials.
when the rostral parts of the MD were stimulated, and was lowest (less than 25/~A) when the rostroventral and relatively lateral part of the MD (MDrvl) was stimulated (Fig. 3B-l, B-2). The paracentral nucleus (PAC) and central lateral nucleus (CLN), which are located rostroventral and rostrolateral to the MD, respectively, were stimulated in two cats. In both cases, stimulation of these nuclei did not evoke any field potentials with such a low threshold as in the case of MD stimulation. Stimulation of the SMT and HL was also ineffective in eliciting any field potentials in the prefrontal and adjacent cortices. Fig. 4 shows schematically the amplitude and distribution of superficial and deep thalamocortical responses evoked by MD stimulation, respectively. Deep thalamocortical responses were large in amplitude in the FRrv, RErd and PROdl (Fig. 4A). Superficial thalamocortical responses were large in the FRrv and RErd (Fig. 4B). Although both deep and superficial thalamocortical responses were prominent in the FRrv and RErd, the deep thalamocortical responses were larger than the superficial ones in the rostroventral part of these areas and the superficial thalamocortical responses were larger than the deep ones in the caudodorsal part. The field potentials could not be examined in the me-
141
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Fig. 5. Distribution of anterogradely labeled terminals following a large injection of HRP into the MD. A: HRP-injection site in the thalamus. Each frontal section of the thalamus is in the same frame as Fig. 3B-l, B-2. B: frontal sections showing HRP-labeled terminals in the prefrontal and adjacent cortices ipsilateral to the HRP-injected thalamus. Photomicrograph of the section indicated by arrow is shown in Fig. 8. The density of the labeled terminals is shown in 3 grades. Denser dots mean a higher density in the terminals.
dial bank of the PRS (PRSm), since it was technically difficult to expose the P R S m and to introduce the recording electrode perpendicularly to the surface.
Anatomy There were 15 cases in which the HRP-injection sites were mostly or exclusively in the MD; three such cases are illustrated in Figs. 5-7. Fig. 8 shows the microphotograph obtained from the case illustrated in Fig. 5B. Fig. 5 shows the distribution of anterogradely labeled
terminals in the prefrontal and adjacent cortices following a large H R P injection into the MD. The injected H R P covered most parts of the MD, except the medial part. It spread to the adjacent nuclei such as the CLN, P A C , VMP, V B A and V L in rostral sections (Fig. 5A). Labeled terminals in both the superficial layer (the outer half of layer I) and the deep layers (from the inner half of layer I I I to the outer half of layer V) were found in the FR, R E , Clant, P R O and PRSm (Fig. 5B). They became sparser in the more caudal areas. In the deep
142
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Fig. 6. Distribution of anterogradely labeled terminals in the prefrontal and adjacent cortices, following a small injection of HRP into the medial part of the MD. A: HRP-injeetion site in the thalamus. B: HRP-labeled terminals in the prefrontal and adjacent cortices shown in the same manner as in Fig. 5B.
layers, the terminals were particularly dense in layer IV of the granular cortex. The dense teminals in layer I were found in the following areas: the FRrv, RErd, P R O and PRSm. In the rostral part of the prefrontal cortex, the dense terminals in layers I I I - V were distributed in almost the same areas as those in layer I. In the caudodorsal part of the F R (FRcd) and the Clant, the labeled terminals in layer I were denser than those in layers I I I - V . In the caudal part of the PRSm, the labeled terminals in layers I I I - V were much denser than those in layer I.
Fig. 6 shows the distribution of labeled terminals following H R P injection into the medial part of the MD. The injected H R P spread slightly to the RH. Labeled terminals were only found in the RE. The labeled terminals were distributed separately in the outer half of layer I, and from the inner half of layer III to the outer half of layer V, in the same manner as in Fig. 5. The labeled terminals in layers I I I - V were denser than those in layer I in the rostroventral part of the RE. Fig. 7 shows the distribution of anterogradely labeled terminals when the injected H R P covered the MDrvl.
143
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Fig. 7. Distribution of anterogradely labeled terminals when injected HRP covered the MDrvl (the rostroventral and relatively lateral part of the MD). A: the HRP-injection site in the thalamus. B: HRP-labeled terminals in the prefrontal and adjacent cortices shown in the same manner as in Fig. 5B.
The MDrvl is the part of the MD where the stimulating electrodes elicited field potentials with the lowest thresholds (less than 25 ~A) in the prefrontal cortex (see Fig. 3B-l, B-2). The injected HRP extended slightly to the LD, PAC and CLN. Densely labeled terminals in layer I were in the FRrv and RErd. Densely labeled terminals in layers I I I - V were in the FRrv, RErd, PROdl and PRSm. Although the amount of H R P injected was rather small, there were very densely labeled terminals in the prefrontal cortex. In addition to the cases in Figs. 5-7, the present study
obtained the following results. When HRP was injected into the most lateral part of the MD, labeled terminals were found only in the dorsal parts of the PRO and PRSm. In the cases where the HRP injection was made selectively in the intermediate part of the MD, labeled terminals were abundant in the mesial region of the prefrontal and adjacent cortices, but rather sparse in their lateral region. In particular, densely labeled terminals were observed in the ventromedial part of the mesial region. In all 15 cases where the HRP was injected into the
144
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III
IV V
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Fig. 8. Photomicrograph of the anterogradely labeled terminals in the coronal section of the prefrontal cortex. A-P level of this section is indicated by the arrow in Fig. 5B. Roman numerals signify the cortical layers. This section was unstained and photographed under dark-field illumination. Bar = 200 pm.
MD, labeled terminals were distributed in the FR, RE, CIant, PRO and PRSm. Injected H R P was confined within the MD in 5 of the 15 cases, in which labeled terminals were found in the same cortical areas as mentioned above. These cortical areas coincide with the areas where field potentials were elicited, except in the PRSm in which field potentials could not be examined. There were no labeled terminals in these cortical areas when H R P was injected into various nuclei (PAC, CLN, SMT, HL, RH) surrounding the MD. On the whole, the labeled terminals had the highest densities in almost the same areas in which the field potentials were elicited with the lowest threshold. DISCUSSION There have been some morphological studies on thalamocortical projections from the MD to the prefrontal cortex in the cat, using the HRP-retrograde transport method 14ASA9. Markowitsch et al. 14 found that the MD projected to the PRO, both banks of the PRS, the ventral part of the gyrus sigmoideus anterior, the rostral and
middle parts of the FR, and the RE. In the present anatomical study using the HRP-anterograde axonal transport method, almost all parts of the MD were injected with H R P (Fig. 5A and 6A). There were, however, no findings that MD neurons project to the ventral part of the gyrus sigmoideus anterior and to the lateral bank of the PRS. The present electrophysiological and anatomical results demonstrated that MD neurons project to the FR, RE, CIant and PRO in the prefrontal and adjacent cortices (see Figs. 3, 5 and 6). Concerning the limits of the projection areas, these results coincide largely with the anatomical findings by other authors 15'~9. Those authors reported that MD fibers projecting to the prefrontal cortex terminate in the FR, RE, PRO and PRSm. The present study demonstrated the projections from the MD to the area 6 in the dorsal part of the FR, and to the CIant, using both electrophysiological and anatomical methods. These findings support the previous reports of following workers. Olson and Jeffers 2~ reported that MD neurons project to the dorsal part of the FR (area 6). It was reported that MD neurons project to the CIant 4'6, premotor cortex 9'~7 and SMA 4'6'18, in addition to the prefrontal cortex, by H R P axonal transport studies in the monkey. The present electrophysiological and anatomical results demonstrate that the whole rostrocaudal extent of the MD projects to the prefrontal cortex. This finding agrees with that of Martinez-Moreno et al. tS. In addition to the fact noted above, the denser thalamocortical projections to the prefrontal cortex are found from the more rostral parts of the MD, i.e., from the MDrvl to the PROdl, FRrv and RErd. There has been an anatomical report, made by Giguere and Goldman-Rakic 4, that MD fibers terminate in layer IV and the adjacent deep portion of layer III, but not at all in layer I, in the prefrontal and adjacent cortices of the monkey. The distribution of MD-prefrontal projection fibers in different cortical layers has, however, not yet been reported in the cat. The present studies first demonstrated that MD fibers terminate in both layer I and layers I I I - V in the prefrontal and adjacent cortices. The differences between their results and the present ones might be the species differences. The labeled terminals from the inner half of layer III to the outer half of layer V were distributed in the prefrontal and adjacent cortical areas where the deep thalamocortical responses were elicited. Such terminals were dense in the FRrv, RErd and PROdl, where the deep thalamocortical responses were prominent. The labeled terminals in the outer half of layer I were distributed in the prefrontal and adjacent cortical areas where the superficial thalamocortical responses were elicited. The terminals were dense in the FRrd and RErv, where
145 the superficial thalamocortical responses were promi-
ods. Therefore, the field potentials elicited by M D stim-
nent. These results revealed that both the superficial and
ulation could not be due to current spread to the thalamic
deep thalamocortical responses in the prefrontal and ad-
nuclei surrounding the MD, and to the structures beyond. D e n s e thalamocortical projections from the V M P in
jacent cortices are due to the thalamocortical projection fibers terminating in layer I and layers I I I - V , respectively. The present studies provide electrophysiological and anatomical evidence for thalamocortical projections from the M D to the prefrontal and adjacent cortices. There were found no thalamocortical projections from the thalamic nuclei surrounding the M D such as the P A C , CLN, SMT and HL, to the prefrontal and adjacent cortices, by either electrophysiological or anatomical meth-
addition to projections from the M D to the prefrontal and adjacent cortices were also demonstrated by both electrophysiological and anatomical studies, as have been reported by G l e n n et al. 5 (Tanibuchi, in preparation).
Acknowledgements. The author wishes to thank Prof. K. Sasaki and Dr. K. Jinnai for their encouragement, helpful advice and critical reading of the manuscript.
ABBREVIATIONS
VMB VMP
basal ventromedial nucleus principal ventromedial nucleus
Thalamus CLN CM CMN HL LD LP MD PAC PF RH SMT SUM VBA VL
Cortex CIant CRU FR FRrv PRO PROd PROdl PROv PRS PRSm RE RErd
gyms cinguli anterior sulcus cruciatus gyrus frontalis rostroventral part of FR gyms proteus dorsal part of PRO dorsolateral part of PRO ventral part of PRO sulcus presylvius medial bank of PRS gyms rectus rostrodorsal part of RE
central lateral nucleus centre median nucleus central medial nucleus lateral habenular nucleus lateral dorsal nucleus lateral posterior complex mediodorsal nucleus paracentral nucleus parafascicular nucleus rhomboid nucleus stria medullaris thalami submedial nucleus ventrobasal complex, arcuate nucleus ventrolateral complex
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28
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Exp. Brain Res., 16 (1972) 89-103. 29 Sasaki, K., Matsuda, Y., Oka, H. and Mizuno, N., Thalamocortical projections for recruiting responses and spindling-like responses in the parietal cortex, Exp. Brain Res., 22 (1975) 8796. 30 Sasaki, K., Staunton, H.P. and Dieckmann, G., Characteristic features of augmenting and recruiting responses in the cerebral cortex, Exp. Neurol., 26 (1970) 369-392. 31 Shinoda, Y. and Kakei, S., Distribution of terminals of thalamocortical fibers originating from the ventrolateral nucleus of the cat thalamus, Neurosci. Lett., 96 (1989) 163-167. 32 Spencer, W.A. and Brookhart, J.M., Electrical patterns of augmenting and recruiting waves in depths of sensorimotor cortex of cat, J. Neurophysiol., 24 (1961) 26-49. 33 Streit, P. and Reubi, J.C., A new and sensitive staining method for axonally transported horseradish peroxidase (HRP) in the pigeon visual system, Brain Res., 126 (1977) 530-537. 34 Tanabe, T., Yarita, H., Iino, M., Ooshima, Y. and Takagi, S.F., An olfactory projection area in orbitofrontal cortex of the monkey, J. Neurophysiol., 38 (1975) 1269-1283. 35 Yarita, H., Iino, M., Tanabe, T., Kogure, S. and Takagi, S.F., A transthalamic olfactory pathway to orbitofrontal cortex in the monkey, J. Neurophysiol., 43 (1980) 69-85. 36 Zola-Morgan, S. and Squire, L.R., Amnesia in monkeys after lesions of the mediodorsal nucleus of the thalamus, Ann. Neurol., 17 (1985) 558-564.
137
BRES 17716
Electrophysiological and anatomical studies on thalamic mediodorsal nucleus projections onto the prefrontal cortex in the cat Ikuo Tanibuchi Department of Integrative Brain Science, Faculty of Medicine, Kyoto University, Kyoto (Japan) (Accepted 24 December 1991) Key words: Thalamocortical projection; Mediodorsal nucleus; Prefrontal cortex; Field potential; Horseradish peroxidase
Electrical stimulation of the mediodorsal nucleus (MD) of the thalamus elicited field potentials in the gyrus proreus (PRO), frontalis (FR), rectus (RE) and cinguli anterior (CIant) of the ipsilateral prefrontal and adjacent cortical areas in cats. The results of a laminar field potential analysis indicate that the field potentials can be regarded as a combination of deep and superficial thalamocortical responses. By injecting horseradish peroxidase (HRP) into the MD, HRP-labeled terminals were distributed in the prefrontal and adjacent cortical areas where the field potentials were elicited. Densely labeled terminals in cortical layer I were distributed where the superficial thalamocortical responses were prominent, while those in layers III-V were distributed in the areas where the deep thalamocortical responses were prominent. INTRODUCTION It is generally thought that the mediodorsal nucleus (MD) of the thalamus, in connection with the prefrontal cortex, plays an important role in learning, memory and olfactionl,13, 24,34-36. The frontal cortical areas where MD neurons project were defined as the prefrontal cortex by Rose and Woolsey 23 ('orbitofrontal cortex' in their terminology). Anatomical studies in the rat, cat and monkey, however, disclosed that the MD also projects to cortical areas other than the prefrontal cortex 4'6'9'1°'17'18'2°'21. Because of the disagreement among the morphological studies on the MD-prefrontal projections 14'15'19, the precise boundary of the prefrontal cortex has not yet been established in the cat. There are also some differences among these morphological findings in the topography of the thalamocortical projections from the subdivisions of the MD to the prefrontal cortex. The first aim of the present study was to determine the MD projection areas in the prefrontal and adjacent cortical areas of cats with both electrophysiological and morphological methods, i.e., an analysis of cortical field potentials elicited by MD stimulation, and anterograde axonal transport study after horseradish peroxidase (HRP) injection into the MD. Thalamocortically evoked potentials in the motor and sensory cortices, as well as in the parietal association
cortex have been investigated in cats 11'12'16'25-30'32. According to an analysis of laminar field potentials, thalamocortically evoked potentials in these cortical areas were found to consist of two kinds of elementary responses, superficial and deep thalamocortical potentials 3°. Superficial thalamocortical responses are surface negative-depth positive (sN-dP) potentials, due mainly to excitatory postsynaptic currents (EPSCs), generated by thalamocortical afferent fibers activating superficial parts of the apical dendrites of pyramidal neurons. The surface negative potentials usually turn positive at a depth of 0.2 mm. Thalamocortical afferents reaching layer I directly are considered to be the morphological substrates of the superficial thalamocortical projections 7' 8,31. Deep thalamocortical responses are surface positive-depth negative (sP-dN) potentials, which usually turn negative at a depth of 0.25 mm. They can be accounted for by the EPSCs, which are generated by thalamic afferents making synapses on deep parts of the apical dendrites and the somata of pyramidal neurons. Thalamocortical afferents terminating in layers I I I - V of the cerebral cortex may correspond to these deep thalamocortical projections 7'8'26'30'31. Thalamocortical responses in the prefrontal and adjacent cortices evoked by MD stimulation have not yet been studied in terms of the superficial and deep thalamocortical responses. Thus, the second aim of the present study was to examine whether the two elementary responses, the superfi-
Correspondence: I. Tanibuchi, Department of Integrative Brain Science, Faculty of Medicine, Kyoto University, Yoshidakonoe-cho, Sakyoku, 606, Kyoto, Japan.
138 cial and deep responses, can be attributed to the thalamocortical projection fibers which terminate in layer I and layers III-V, respectively, of the prefrontal and adjacent cortices.
A
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MATERIALS AND METHODS
Electrophysiology Thirty-six adult cats of both sexes, weighing 2.5-5.2 kg, were anesthetized by sodium pentobarbital (30 mg/kg, i.v.) and fixed to a stereotaxic instrument. A craniotomy was performed to expose the lateral or mesial surface of the frontal cortex. These lateral and mesial surfaces were examined in separate experiments. For the experiments examining the mesial surface, the frontal cortex in one side was aspirated out to expose the mesial surface of the contralateral prefrontal cortex. Six to nine concentric, stimulating electrodes (o.d. 0.5 ram, the central core extruding by about 0.5 ram) were introduced vertically into the MD ipsilateral to the exposed prefrontal cortex. The MD was then stimulated at 1 Hz, with a current pulse of 300/~s duration and less than 120/~A. The stimulation sites were examined histologically after every experiment. A glass microelectrode of 2-4 Mf~ DC-resistance filled with 0.75 M sodium acetate (CH3COONa) was inserted into the prefrontal and adjacent cortices perpendicularly to their surface, to record the field potentials. This microelectrode was advanced step by step to a depth of 2.5 mm while field potentials were recorded at various depths. The exposed surface of the cortex was constantly bathed in warm Ringer's solution to protect the cortex from cooling and drying. The body temperature was maintained between 36 and 37°C throughout the surgical and recording procedures.
Anatomy Thirty-two adult cats of both sexes, weighing 2.6-5.1 kg, were used for the HRP study. Under sodium pentobarbital anesthesia (30 mg/kg, i.v.), craniotomies were done in the cats. Wheat germ agglutinin--horseradish peroxidase (WGA-HRP) was injected into the unilateral MD in order to label the terminals of the thalamocortical projection fibers in the ipsilateral prefrontal and adjacent cortices by anterograde axonal transport. The injections of 5% W G A - H R P dissolved in 0.9% saline were made stereotaxically, by using a 1 /~1 Hamilton microsyringe; the volume varied between 0.02 and 0.06/~1 per injection. Following the survival time of 28-48 h, the animals were deeply anesthetized with sodium pentobarbital. They were then perfused intracardially with 500-750 ml of 7% formalin in 0.1 M phosphate buffer (pH 7.6), followed by 0.1 M phosphate buffer containing 10% sucrose. The brains were removed immediately after the perfusion and soaked in 0.1 M phosphate buffer containing 30% sucrose at 4°C for 3-5 days. The frozen brains were then cut serially into frontal sections of 70/~m thickness through the thalamic injection sites and the prefrontal and adjacent cortices. Sections of the thalamus were treated with 3,3'-diaminobenzidine-tetrahydrochloride (DAB) according to the method of Streit and Reubi 33. They were counterstained with cresyl violet, and then examined microscopically under bright-field illumination. The injection site was defined as the region in which dense HRP reaction product was visible under bright-field illumination. Thalamic injection sites were reconstructed using an overhead projector. The sections from the prefrontal and adjacent cortices were treated with benzidine dihydrochloride (BDHC) according to the method of DeOlmos and Heimer 3. They were examined microscopically under dark-field illumination without counterstaining in order to avoid fading of the labeled terminals, and were then counterstained with neutral red in order to reconstruct the laminar structure of the cortex. The distribution of the labeled terminals was charted on the drawings taken from the counterstained sections. Nomenclature and delineation of the thalamic nuclei followed the atlas by Berman and Jones2.
B
Fig. I. Diagrams showing the subdivisions of the prefrontal and adjacent cortices (PRO, PRSm, FR, RE and CIant), based on the atlas by Reinoso-Suarez22. A: the lateral and mesial views of the cat cortex. B: the frontal sections corresponding to the six arrows in each view in A. The same frames are used in Figs. 5-7B.
The subdivisions of the prefrontal and adjacent cortices, used in the following sections, are shown in Fig. 1.
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Fig. 2. Examples of laminar field potentials recorded at various depths (0.0-2.5 mm) from the surface at a, b and c, as indicated in Fig. 3. About 6 sweeps were superimposed on each trace. The depth for column b is the same as for a. a: deep thalamocortical response, b: a succession of deep and superficial thalamoeortical responses, c: superficial thalamocortical response. Voltage calibration for columns a and b is 1 mV, and is 0.5 mV for column c. The time scale for all records is 50 ms. Depths are indicated in millimeters on the left of a and c.
139 threshold deep+superficial •
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Fig. 3. A-1 and A-2 show the distribution of the field potentials which MD stimulation elicited in the mesial and lateral regions of the prefrontal and adjacent cortices, respectively. Open circles, filled squares and filled circles show deep, superficial and a succession of deep and superficial thalamocortical responses, respectively. Their sizes are inversely proportional to the thresholds of the field potentials elicited, i.e, large marks show field potentials with low thresholds. Crosses show no response. B-1 and B-2 show the stimulation sites in the MD which evoked field potentials in the mesial and lateral regions of the prefrontal and adjacent cortices, respectively. The frontal sections of the thalamus through the MD (+10.2, 9.5, 9.1, 8.3, 7.9) were ipsilateral to the prefrontal cortex where the field potentials were recorded. The number above each coronal section (right side; medial) indicates the distance from the interaural plane. Arrows accompanied by asterisks indicate the stimulation sites in the MD (MDrvl) which evoked the field potentials with the lowest threshold.
140 RESULTS
Electrophysiology Fig. 2 shows examples of field potentials recorded from microelectrode tracks at sites a, b and c in the prefrontal and adjacent cortices (see Fig. 3A-l, A-2). In column a, a single stimulation of the MD produced surface positive (0.00-0.25 mm) and depth negative (0.252.50 mm) (sP-dN) potentials. The reversal of polarity was at a depth of about 0.25 mm. The latency was about 2.5 ms. These characteristics coincide with those of the deep thalamocortical response. The field potentials in column c have the same characteristics as the superficial thalamocortical response. The reversal level was at a depth of about 0.2 mm, and the latency was about 17.8 ms. The evoked potentials in column b can be interpreted as a succession of deep and superficial thalamocortical responses. The superficial thalamocortical response is slightly larger than the deep thalamocortical response. The reversal level of the deep thalamocortical response was at a depth of about 0.25 mm, whereas that of the superficial thalamocortical response apppears to be slightly more superficial than 0.25 mm. The latencies of the deep and superficial thalamocortical responses were about 9.3 ms and about 22.0 ms, respectively. Fig. 3 A-1 and A-2 show the distribution of the field potentials elicited in the prefrontal and adjacent cortical areas by MD stimulation with a current of less than 120 /~A. Field potentials were elicited in the gyrus frontalis (FR) (including area 6), gyrus rectus (RE), gyrus cinguli anterior (Clant) (paralimbic region) and the gyrus proreus (PRO). In most electrode tracks, the initial positive potentials followed by the negative ones were recorded at the surface. The surface positive potentials turned negative at a depth of about 0.25 mm, while the surface negative potentials turned positive at a depth of about 0.20 mm. Most potentials evoked by MD stimulation were therefore regarded as deep thalamocortical responses followed by superficial thalamocortical responses. Pure deep thalamocortical responses were elicited in the rostral part of the RE (REr), and the dorsolateral and ventral parts of the PRO (PROdl and PROv). Pure superficial thalamocortical responses were elicited in the caudodorsal part of the FR (FRcd), and the Clant. The latencies of the deep and superficial thalamocortical responses were 1-16 ms (mean: 6.3 ms), and 14-25 ms (mean: 18.8 ms), respectively. The field potentials in the mesial prefrontal and adjacent cortices (FR, RE) were evoked by stimulation of the whole rostrocaudal extent of the MD (Fig. 3B-l), while +hose in the lateral prefrontal cortex (PRO) were evoked by stimulation of the rostral part of the MD (Fig. 3B-2). The threshold for eliciting field potentials tended to be low
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Fig. 4. Distribution of the deep thalamocortical (filled circles in A) and superficial thalamocortical (filled squares in B) responses in the prefrontal and adjacent cortical areas of the cat, evoked by MD stimulation with a current of 120 ~A. Their relative sizes are in proportion to the amplitude of the field potentials.
when the rostral parts of the MD were stimulated, and was lowest (less than 25/~A) when the rostroventral and relatively lateral part of the MD (MDrvl) was stimulated (Fig. 3B-l, B-2). The paracentral nucleus (PAC) and central lateral nucleus (CLN), which are located rostroventral and rostrolateral to the MD, respectively, were stimulated in two cats. In both cases, stimulation of these nuclei did not evoke any field potentials with such a low threshold as in the case of MD stimulation. Stimulation of the SMT and HL was also ineffective in eliciting any field potentials in the prefrontal and adjacent cortices. Fig. 4 shows schematically the amplitude and distribution of superficial and deep thalamocortical responses evoked by MD stimulation, respectively. Deep thalamocortical responses were large in amplitude in the FRrv, RErd and PROdl (Fig. 4A). Superficial thalamocortical responses were large in the FRrv and RErd (Fig. 4B). Although both deep and superficial thalamocortical responses were prominent in the FRrv and RErd, the deep thalamocortical responses were larger than the superficial ones in the rostroventral part of these areas and the superficial thalamocortical responses were larger than the deep ones in the caudodorsal part. The field potentials could not be examined in the me-
141
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Fig. 5. Distribution of anterogradely labeled terminals following a large injection of HRP into the MD. A: HRP-injection site in the thalamus. Each frontal section of the thalamus is in the same frame as Fig. 3B-l, B-2. B: frontal sections showing HRP-labeled terminals in the prefrontal and adjacent cortices ipsilateral to the HRP-injected thalamus. Photomicrograph of the section indicated by arrow is shown in Fig. 8. The density of the labeled terminals is shown in 3 grades. Denser dots mean a higher density in the terminals.
dial bank of the PRS (PRSm), since it was technically difficult to expose the P R S m and to introduce the recording electrode perpendicularly to the surface.
Anatomy There were 15 cases in which the HRP-injection sites were mostly or exclusively in the MD; three such cases are illustrated in Figs. 5-7. Fig. 8 shows the microphotograph obtained from the case illustrated in Fig. 5B. Fig. 5 shows the distribution of anterogradely labeled
terminals in the prefrontal and adjacent cortices following a large H R P injection into the MD. The injected H R P covered most parts of the MD, except the medial part. It spread to the adjacent nuclei such as the CLN, P A C , VMP, V B A and V L in rostral sections (Fig. 5A). Labeled terminals in both the superficial layer (the outer half of layer I) and the deep layers (from the inner half of layer I I I to the outer half of layer V) were found in the FR, R E , Clant, P R O and PRSm (Fig. 5B). They became sparser in the more caudal areas. In the deep
142
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Fig. 6. Distribution of anterogradely labeled terminals in the prefrontal and adjacent cortices, following a small injection of HRP into the medial part of the MD. A: HRP-injeetion site in the thalamus. B: HRP-labeled terminals in the prefrontal and adjacent cortices shown in the same manner as in Fig. 5B.
layers, the terminals were particularly dense in layer IV of the granular cortex. The dense teminals in layer I were found in the following areas: the FRrv, RErd, P R O and PRSm. In the rostral part of the prefrontal cortex, the dense terminals in layers I I I - V were distributed in almost the same areas as those in layer I. In the caudodorsal part of the F R (FRcd) and the Clant, the labeled terminals in layer I were denser than those in layers I I I - V . In the caudal part of the PRSm, the labeled terminals in layers I I I - V were much denser than those in layer I.
Fig. 6 shows the distribution of labeled terminals following H R P injection into the medial part of the MD. The injected H R P spread slightly to the RH. Labeled terminals were only found in the RE. The labeled terminals were distributed separately in the outer half of layer I, and from the inner half of layer III to the outer half of layer V, in the same manner as in Fig. 5. The labeled terminals in layers I I I - V were denser than those in layer I in the rostroventral part of the RE. Fig. 7 shows the distribution of anterogradely labeled terminals when the injected H R P covered the MDrvl.
143
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Fig. 7. Distribution of anterogradely labeled terminals when injected HRP covered the MDrvl (the rostroventral and relatively lateral part of the MD). A: the HRP-injection site in the thalamus. B: HRP-labeled terminals in the prefrontal and adjacent cortices shown in the same manner as in Fig. 5B.
The MDrvl is the part of the MD where the stimulating electrodes elicited field potentials with the lowest thresholds (less than 25 ~A) in the prefrontal cortex (see Fig. 3B-l, B-2). The injected HRP extended slightly to the LD, PAC and CLN. Densely labeled terminals in layer I were in the FRrv and RErd. Densely labeled terminals in layers I I I - V were in the FRrv, RErd, PROdl and PRSm. Although the amount of H R P injected was rather small, there were very densely labeled terminals in the prefrontal cortex. In addition to the cases in Figs. 5-7, the present study
obtained the following results. When HRP was injected into the most lateral part of the MD, labeled terminals were found only in the dorsal parts of the PRO and PRSm. In the cases where the HRP injection was made selectively in the intermediate part of the MD, labeled terminals were abundant in the mesial region of the prefrontal and adjacent cortices, but rather sparse in their lateral region. In particular, densely labeled terminals were observed in the ventromedial part of the mesial region. In all 15 cases where the HRP was injected into the
144
II
III
IV V
VI
Fig. 8. Photomicrograph of the anterogradely labeled terminals in the coronal section of the prefrontal cortex. A-P level of this section is indicated by the arrow in Fig. 5B. Roman numerals signify the cortical layers. This section was unstained and photographed under dark-field illumination. Bar = 200 pm.
MD, labeled terminals were distributed in the FR, RE, CIant, PRO and PRSm. Injected H R P was confined within the MD in 5 of the 15 cases, in which labeled terminals were found in the same cortical areas as mentioned above. These cortical areas coincide with the areas where field potentials were elicited, except in the PRSm in which field potentials could not be examined. There were no labeled terminals in these cortical areas when H R P was injected into various nuclei (PAC, CLN, SMT, HL, RH) surrounding the MD. On the whole, the labeled terminals had the highest densities in almost the same areas in which the field potentials were elicited with the lowest threshold. DISCUSSION There have been some morphological studies on thalamocortical projections from the MD to the prefrontal cortex in the cat, using the HRP-retrograde transport method 14ASA9. Markowitsch et al. 14 found that the MD projected to the PRO, both banks of the PRS, the ventral part of the gyrus sigmoideus anterior, the rostral and
middle parts of the FR, and the RE. In the present anatomical study using the HRP-anterograde axonal transport method, almost all parts of the MD were injected with H R P (Fig. 5A and 6A). There were, however, no findings that MD neurons project to the ventral part of the gyrus sigmoideus anterior and to the lateral bank of the PRS. The present electrophysiological and anatomical results demonstrated that MD neurons project to the FR, RE, CIant and PRO in the prefrontal and adjacent cortices (see Figs. 3, 5 and 6). Concerning the limits of the projection areas, these results coincide largely with the anatomical findings by other authors 15'~9. Those authors reported that MD fibers projecting to the prefrontal cortex terminate in the FR, RE, PRO and PRSm. The present study demonstrated the projections from the MD to the area 6 in the dorsal part of the FR, and to the CIant, using both electrophysiological and anatomical methods. These findings support the previous reports of following workers. Olson and Jeffers 2~ reported that MD neurons project to the dorsal part of the FR (area 6). It was reported that MD neurons project to the CIant 4'6, premotor cortex 9'~7 and SMA 4'6'18, in addition to the prefrontal cortex, by H R P axonal transport studies in the monkey. The present electrophysiological and anatomical results demonstrate that the whole rostrocaudal extent of the MD projects to the prefrontal cortex. This finding agrees with that of Martinez-Moreno et al. tS. In addition to the fact noted above, the denser thalamocortical projections to the prefrontal cortex are found from the more rostral parts of the MD, i.e., from the MDrvl to the PROdl, FRrv and RErd. There has been an anatomical report, made by Giguere and Goldman-Rakic 4, that MD fibers terminate in layer IV and the adjacent deep portion of layer III, but not at all in layer I, in the prefrontal and adjacent cortices of the monkey. The distribution of MD-prefrontal projection fibers in different cortical layers has, however, not yet been reported in the cat. The present studies first demonstrated that MD fibers terminate in both layer I and layers I I I - V in the prefrontal and adjacent cortices. The differences between their results and the present ones might be the species differences. The labeled terminals from the inner half of layer III to the outer half of layer V were distributed in the prefrontal and adjacent cortical areas where the deep thalamocortical responses were elicited. Such terminals were dense in the FRrv, RErd and PROdl, where the deep thalamocortical responses were prominent. The labeled terminals in the outer half of layer I were distributed in the prefrontal and adjacent cortical areas where the superficial thalamocortical responses were elicited. The terminals were dense in the FRrd and RErv, where
145 the superficial thalamocortical responses were promi-
ods. Therefore, the field potentials elicited by M D stim-
nent. These results revealed that both the superficial and
ulation could not be due to current spread to the thalamic
deep thalamocortical responses in the prefrontal and ad-
nuclei surrounding the MD, and to the structures beyond. D e n s e thalamocortical projections from the V M P in
jacent cortices are due to the thalamocortical projection fibers terminating in layer I and layers I I I - V , respectively. The present studies provide electrophysiological and anatomical evidence for thalamocortical projections from the M D to the prefrontal and adjacent cortices. There were found no thalamocortical projections from the thalamic nuclei surrounding the M D such as the P A C , CLN, SMT and HL, to the prefrontal and adjacent cortices, by either electrophysiological or anatomical meth-
addition to projections from the M D to the prefrontal and adjacent cortices were also demonstrated by both electrophysiological and anatomical studies, as have been reported by G l e n n et al. 5 (Tanibuchi, in preparation).
Acknowledgements. The author wishes to thank Prof. K. Sasaki and Dr. K. Jinnai for their encouragement, helpful advice and critical reading of the manuscript.
ABBREVIATIONS
VMB VMP
basal ventromedial nucleus principal ventromedial nucleus
Thalamus CLN CM CMN HL LD LP MD PAC PF RH SMT SUM VBA VL
Cortex CIant CRU FR FRrv PRO PROd PROdl PROv PRS PRSm RE RErd
gyms cinguli anterior sulcus cruciatus gyrus frontalis rostroventral part of FR gyms proteus dorsal part of PRO dorsolateral part of PRO ventral part of PRO sulcus presylvius medial bank of PRS gyms rectus rostrodorsal part of RE
central lateral nucleus centre median nucleus central medial nucleus lateral habenular nucleus lateral dorsal nucleus lateral posterior complex mediodorsal nucleus paracentral nucleus parafascicular nucleus rhomboid nucleus stria medullaris thalami submedial nucleus ventrobasal complex, arcuate nucleus ventrolateral complex
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