1% Showed Wide Receptive Fields, Or Bilat- Eral Representation

Somatotopic Organization and Response Properties of Neurons of the Ventrobasal Complex in the Opossum '

AGLAI P. B. SOUSA,Z E. OSWALDO-CRUZ AND R. GATTASS 2 Departamento de Neurobiologia, Institvto de Bioflsica, 458 av. Pasteur, Rio de Janeiro, Brad

ABSTRACT Thalamic responses to somatic stimulation were studied in 47 specimens of the South American opossum, D. marsupialis aurita. Topographic representation of the body surface in the ventrobasal complex was determined by recording unit cluster responses to natural stimulation in sodium pentobarbital anesthetised animals. The pattern of representation observed was similar to that reported in the North American subspecies and comparable to the findings in eutherian mammals. The analysis of single units recorded with extra-cellular microelectrodes showed that the great majority of neurons are place and modality specific. A predominance of skin (vs. deep) sensibility was observed. Units activated by displacement of hairs and vibrissae displayed receptive fields similar to those reported in higher mammals, Of the units isolated within the anatomical boundaries of the ventrobasal complex only 1% showed wide receptive fields, or bilateral representation. None of the units could be activated by auditory stimulation. Neurons displaying wide, discontinuous or bilateral receptive fields showing auditory-somatic convergence were isolated in nucleus paraf ascicularis, subparafascicularis and in the Posterior group

Using anatomical techniques it has been nervation density are related to a larger shown, in a wide variety of mammals, that voIume of thalamic tissue. components of the ventrobasal complex Single unit recording from this region in (xVB), as defined by Rose and Mount- cats showed that the neurons receiving castle ('52) are the main site of termina- lemniscal projections are place and modal- tion of the medial lemniscus. Recent ana- ity specific (Rose and Mountcastle, '54). tomical literature related to this problem Poggio and Mountcastle ('63) demon- was reviewed by Bowsher ('65). strated, by recording more than 1,000 units After the introduction of electrophysio- from the xVB in chronically denervated logical recording methods, a precise organi- monkeys, that this restricted input is not zation of the somatic sensory projections due to the use of anesthetic agents. within the anatomical boundaries of this In eutherian mammals results differing from this pattern have been occasionally nucleus was demonstrated in several spe- reported. Besides cortico-dependent neu- cies (rat: Angel, '64; Davidson, '65; Em- rons the ventrobasal complex has been mers, '65; rabbit: Rose and Mountcastle, shown to include elements that survive de- '52; Mountcastle et al., '52; raccoon: cortication (Clark and Powell, '53; Bava Welker and Johnson, '65; sheep: Cabral and Johnson, '71; rhesus monkey: Mount- et al., '66). They possibly represent the in- terneurons described in the posterolateral castle and Henneman, '52; cynomdogus xVB by Andersen et al. ('64). monkey : Albe-Fessard and Bowsher, '65; spider monkey: Pubols, '68). It was shown 1 This investigation was supported by Public Health Service Research grant NB 04651, from the National that the body surface is represented in a Institute of Neurological Diseases and Stroke; BNDE Contract FUNTEC-74 and Conselho de Pesquisas da circumscribed region within the ventro- U.F.R.J. basal complex. In this representation pe- ZFellow from the Conselho Nacional de Pesquisas. 3 Senior Researcher (Pesquisador Conferencista) ripheral regions displaying a greater in- Conselho Nacional de Pesquisas.

J. COMP. NEUR., 142: 231-248. 23 1 232 A. P. B. SOUSA, E. OSWALDOCRUZ AND R. GATTASS

By determining the site of termination neuroanatomical investigations (for a com- of the spinothalamic tract, it was shown plete review see Oswaldo-Cruz and Rocha- that the ventrobasal complex is not the Miranda, '68). The morphological features only region receiving somatic projections. of the opossum thalamus, which have been The small calibre fibers of this tract were described by various authors (Bodian, shown to project to a wider area, which '39; Diamond and Utley, '63; Oswaldo- includes the caudal pole of the ventrobasal Cruz and Rocha-Miranda, '67a,b, '68), are complex, the posterior group (Po, as de- similar to those of higher mammals. fined by Rose and Woolsey, '58), the para- Recording from single units in the xVB fascicular-center median region, as well of the North American subspecies, Erick- as some of the intralaminar nuclei (Mehler son et al. ('64) reported a large percentage et al., '60). of units displaying wide receptive fields Single unit analysis of the Po region and convergence of sensory modalities. in the cat (Poggio and Mountcastle, '60; This result would support the idea that the Whitlock and Perl, '59; '61) shows that the metatheria display a primitive form of functional properties of its component thalamic organization, intermediate be- cells are different from those reported in tween the reptilian and the higher mam- the ventrobasal complex. The projections malian pattern. On the other hand, using to this region are attributed to the antero- the same species but recording unit cluster lateral spinal tract. A large percentage of responses to tactile stimulation, Pubols its component cells display not only wide and Pubols ('66) reported a precise somato- receptive fields, often bilateral, but also topical organization within the boundaries convergence of different sensory modal- of the xVB, similar in all respects to the ities. results obtained in eutherian mammals. Similar properties and functional organi- In view of the conflicting results ob- zation were found in the cortical sensory tained in the study of the opossum thala- areas; SI displaying properties akin to mus, the following work was carried out to those of the ventrobasal complex (Mount- clarify the points in variance. castle et al., '57; Mountcastle and Powell, The functional organization of the xVB '59; Powell and Mountcastle, '59), while was studied in a South American sub- SII can be related to the properties dis- species (D. marsupialis aurita Wied, 1826). played by Po (Andersson, '62; Carreras and In this species the cytoarchitectonic organi- Andersson, '63; Berman, '61a,b). All of zation of the central nervous system has the above results were obtained in various been shown to be identical to that of the species of eutherian mammals. North American variety by detailed ana- The functional properties displayed by tomical and statistical studies (Oswaldo- these divisions of the somatic sensory Cruz and Rocha-Miranda, '67a,b, '68). mechanisms conducted by ( 1) the dorsal Our description of the ventrobasal com- column-medial lemniscus and by (2) the plex closely corresponds with Bodian's ('39) anterolateral pathway have been corre- nucleus ventralis pars principalis, except lated by Rose and Mountcastle ('59) with for the possible inclusion of part of our respectively, ( 1) the epicritic and (2) pro- nucleus ventralis thalami lateralis and topathic concepts expressed by Head et nucleus C (see Oswaldo-Cruz and Rocha- al. ('20). Miranda, '67a,b, '68). This complex, ovoid In view of the proposed theories regard- in shape, extends from A 5.50 to A 3.17. ing the evolution of the sensory systems The rostro-caudal axis of this nucleus is and that of the thalamus (Bishop, '59; Dia- ventrally tilted in respect to the Horsley- mond and Chow, '62), it would be of Clarke basal plane. In the medio-lateral interest to extend these studies to primitive direction it attains a maximum width of mammals, representatives of the meta- 2.2 mm at its mid-portion; the dorso-ven- theria group. tral extent reaching its maximum of The common opossum (Didelphis mar- 1.5 mm at the same level. supialis, Linnaeus, 1758), being a species The medial border of this complex is of wide ecological distribution in the con- with nuclei ventralis thalami medialis ex- tinent, has been the subject of numerous cept at its caudal tip, where nuclei subparu- VENTROBASAL COMPLEX OF THE OPOSSUM 233 fascicularis and posterioris thalami re- filled with warm Ringer or mineral oil. The place the latter. The ventro and lateral dura was incised and, in all animals used borders are given by the well developed for microelectrode recording, the cortical lamina medullaris externa, except at pos- gray overlaying the thalamic region under- terior planes where nucleus posterioris going exploration was removed by sub-pial thalami is found capping the caudal pole suction. of the xVB. Dorsally, in its rostral pole, Penetrations were oriented according to the complex is related with nuclei intra- the stereotaxic atlas developed in this lab- laminaris, nucleus lateralis thalami inter- oratory for both the North and South medius and nucleus ventralis thalami Eat- eralis. At a caudal level nucleus C makes American subspecies ( Oswaldo-Cruz and its appearance as the dorsal border reach- Rocha-Miranda, '68). ing the caudal pole of the complex. In four animals the dorsal column and/ The rostral limit of this complex is diffi- or dorsolateral quadrant were sectioned at cult to determine in anatomical grounds, high cervical level. The pia-arachnoid for it gradually merges with nuclei ven- membrane was dissected and the lesion tralis thalami lateralis. accomplished by means of a razor blade fragment, the extent of the lesion being MATERIAL AND METHODS later determined by microscopic inspec- Two experimental series are included in tions of paraffin embedded, Nissl stained the present work. In the first group of 19 frontal sections. animals, unit cluster activity was recorded Recording methods. Unit cluster re- in order to establish the topography of sponses were recorded using steel needles projections of the body surface in the xVB. prepared from dental broaches, approxi- A second group, comprising 28 animals, mately 300 in diameter, showing a grad- was used to study the functional charac- ual taper towards the tip. The electrodes teristics of single units in the xVB and in were varnish-insulated except at the tip, neighboring structures. Included in this where a region of approximately 150- group are four animals in which lesions in 250 was left exposed. the dorsal column and dorsolateral quad- The electrical activity recorded by these rant of the spinal cord were made. electrodes was led, through a Grass high A total of 47 adult specimens of Didel- impedence probe (HP-5), to a high gain phis marsupialis aurita, trapped in the differential amplifier (Grass P5). The in- wilds in and around Riode Janeiro, were different electrode was connected to the used. stereotaxic frame. The electrical activity Preparation of the subject. All animals was displayed in a dual beam Tektronix used for unit cluster recording were anes- 502 oscilloscope and simultaneously fed to thetised with an initial intraperitoneal an audio-monitor system. The characteris- dose of sodium pentobarbital (30 mg/kg), tic change in the xVB background noise, maintenance doses being supplied through resulting from peripheral tactile stimula- an intravenous cannula at a rate of ap- tion, was the main criterion to determine proximately 10mg every 30 minutes. the active region. For single unit recording 21 animals re- Single unit activity was recorded by ceived identical treatment, while five ani- means of indium-filled, gold and platinum mals were anesthetised with Dial-urethane plated electrodes, of the type described by (100-500 mg/kg) and two animals with Dowben and Rose ('53). For the last ex- a-chloralose (80 mg/kg). periments of the microelectrode series the The anesthetised animal had its fur electrodes were prepared by etching fine closely clipped, and following the insertion tungsten wire (Hubel, '57). After gold of tracheal and venous cannulae, the ani- pIating of the tip, the electrodes were mal was fixed to a Horsley-Clarke instru- varnish insulated and oven baked. In order ment specially modified to accommodate to reduce tip impedance platinum black this species (Rocha-Miranda et al., '68). was deposited just before use. The ability A bilateral craniotomy was made, and a of these electrodes to isolate unitary activ- dam of auick setting dental impression ity was comparable to that of the indium compound erected around the opening was type 9 234 A. P. B. SOUSA, E. OSWALDO-CRUZ AND R. GATTASS

The activity recorded by the microelec- switch permitted simultaneous Stimulation trode was fed to a solid state field-effect of all points or restricted the electrical probe, and, after further amplification, to pulse to a selected single point. the display system. Slow activity was fil- Auditory stimulation was accomplished tered at the amplifier stage. A test signal by a short pulse fed to a loud speaker, re- could be introduced through the prepara- sulting in a click sound, or by hand clap- tion at any time during the experiment ping in the immediate vicinity of the ani- to evaluate the electrode impedance. In mal. Visual stimulation was achieved by order to facilitate the isolation of single means of short flashes of a glow modulator units, a differential amplitude discrimina- tube or by mechanical interruption of the tor was incorporated to the system. In addi- light beam of a tungsten filament lamp, tion, interval timers and EPUT meters directed into the eye. Monitoring of visually were used to supplement information con- evoked responses at the superior colliculus, cerning latency values and discharge rate the optic mesencephalic tract, the dorsal of single units isolated by the discrimina- geniculate body, and the pretectal nucleus tor. was used to establish functional reference It is a well established fact that initially points to determine the depth of the pene- negative units are less liable to display trations. signs of injury (Mountcastle et al., ’57). Mapping procedures. In order to study The majority of units were studied while the topographic arrangements in the xVB, displaying negative spikes, and only a macroelectrodes were lowered in steps of small percentage of initially positive units 50-1OOp, and the entire body surface ex- were taken into consideration. plored by natural stimulation, at each step. Stimulation. Natural stimulation was The location and relative intensity of the applied by gentle displacement of hairs evoked response were recorded in a series and vibrissae, by stroking restricted por- of figurines, including detailed views of the tions of the body surface with a camel hair head, hand and pedimanus. The criterion brush or by touching these areas with blunt for establishing the active region was the rods. In order to stimulate deep structures, magnitude of the neural background ac- pressure was applied through the cutane- tivity evoked by natural stimulation, as ous surface, and passive displacement of judged by CRO display and audio-monitor- the joints was carried out manually. “Pro- ing. topathic” projections were tested by prick- Penetrations were made at 0.5mm in- ing with a pin or by pinching the skin with tervals in the mediolateral plane and at jeweler’s forceps. In order to establish 0.75-1.0 mm in the AP plane. Penetrations latency values and responsiveness to repeti- at smaller intervals disrupted the cellular tive stimulation, uninsulated needles were pattern, rendering the nuclear borders un- thrust into the receptive field, and electri- certain. All penetrations, in a given AP cal stimulation was applied through a plane, were lowered to the same horizontal stimulus isolation unit (Grass SIU). In level whenever possible. This precaution, nine animals electrical stimulation was together with reference markings, reduced applied to different points of the body sur- the margin of error in estimating the true face, duplicating the procedure used by recording site. Erickson et al. (’64). Uninsulated needles A mechanical micromanipulator having were inserted bilaterally in the rhinarium, adjustments in all three planes was used to region of the snout vibrissae, palm of the position the microelectrodes, vertical dis- hand and pedimanus. Electrical stimula- placements were carried out at a 1 p reso- tion of the tail was not systematically em- lution. The electrode was slowly advanced ployed. Electrical stimuli consisted of 0.2- until the activity of a single unit was iso- 0.3msec pulses, of amplitude of 20V or lated. Isolation of initially negative units more, delivered at a repetition rate of 0.5- was frequently possible only after resorting 1.0 per second. The uninsulated needles to the differential amplitude discriminator. inserted in the above mentioned body re- After isolation, the unit was assigned a gions were connected to a selector switch code number and its receptive field, as well interposed in the stimulation circuit. This as other functional characteristics, deter- VENTROBASAL COMPLEX OF THE OPOSSUM 235 mined. Units lost before the gathering of vating the neural population shifted to complete data on its functional character- another site. istics, or those showing signs of injury The results of a typical penetration are were dropped. shown in figure 1. The reconstruction of Histological procedures. After comple- the track of this penetration indicated that tion of the experiments, the animals were the electrode traversed the xVB at 2.2 mm deeply anesthetised and perfused with from the midline at the plane A 4.2mm. warm saline followed by liberal amounts When the electrode tip entered the dorsal of Baker's buffered formalin. After an border of the xVB complex, the neural adequate hardening period, the brains were activity was seen to be driven by stimula- blocked, in situ, along the plane of pene- tion of a region extending from the rostral tration, using a special stereotaxically ori- edge of the pinna to its base. In the next ented macrotome (Rocha-Miranda et al., 300 p, the activating region moved towards '65). Serial frontal sections were stained the zygomatic and periorbital regions, with Chroma's Cresylecht Violett. reaching the upper row of vibrissae (H5.9). The 19 brains of the first series were In the following 4OOp the activating site embedded in parafin, the remaining 28 consisted of the region of the mystacial were processed by the frozen technique, vibrissae, gradually shifting from the dor- receiving otherwise identical treatment. Treatment of data. In a preliminary sur- vey of the slides the penetrations were identified and the bottom of the tracks A determined. A reconstruction of each penetration was made based on the enlarged images (10 X-15 X ) of selected sections. Locations of recording sites were established taking into consideration corrective factors described in greater detail in a previous publication ( Oswaldo-Cruz and Kocha-Miranda, '68). The tracks of fine microelectrodes were, at times, exceedingly difficult to determine with precision. This 5:s 5:7 5.6 5.5 5.4 task was greatly facilitated by diagonal illumination as suggested by Poggio and Mountcastle ('60). A better result is obtained by means of low aperture dark field technique. 5.2 Using the above mentioned procedures it was possible to correlate recording sites with functional properties. This correlation 5.1 was achieved by plotting all available data in standard planes based on the atlas for this species (Oswaldo-Cruz and Rocha- Miranda, '68). 115-3 4-9 5.0 RESULTS Fig. 1 Results of a macroelectrode descent into the ventrobasal complex at Horsley-Clarke When an electrode was slowly advanced plane A4.2, L2.2. As the electrode is lowered towards the ventrobasal complex, there the receptive field gradually shifts along the was a characteristic change in the back- head in the caudo-rostral direction, from the pinna to the periorbital region and finally to the ground noise as the recording tip ap- vibrissae. With further advancement of the elec- proached its dorsal border. The background trode (5.4) the receptive field moves abruptly to discharge was then modified by tactile stim- the palmar surface, at the base of the first digit. ulation of a restricted region of the contra- At the ventral border of the xVB the projections lateral body surface. With further advance- are restricted to the tip of digit V. The numbem indicating vertical displacement of the electrode ment of the electrode, in small increments do not correspond to the Horsley-Clarke vertical of 100 p or less, the peripheral region acti- axis scale. 236 A. P. B. SOUSA, E. OSWALDO-CRUZ AND R. GATTASS sal to the ventral rows, near the angle of caudal axis along the M-L axis of the nu- the mouth (H5.5). Further advancement cleus, rostral portions of the body repre- of the electrode resulted in an abrupt sented medially in the thalamus; the axial change of the receptive field, now found to parts of the body are represented at the occupy a restricted region of the palmar dorsal border of the nucleus, its ventral surface (H5.4). At still lower levels, the border being occupied mostly by projec- activating site was found near digits IV at tions from the extremities of the limbs. The H5.1 and V at H5.0, and finally at H4.9 trigeminal division is represented through- it was restricted to the tip of digit V. Fur- out the whole extent of the nuclear com- ther displacement of the recording elec- plex, the remaining body surface is repre- trode resulted in an abrupt change of the sented laterally at the central portion of background, as its tip left the ventrobasal the nucleus. complex. Near the midline, somatically evoked ac- Comparable results were obtained in 87 tivity was restricted to the xVB, and no penetrations traversing the xVB. The re- response could be obtained in its medial sults of 34 of these penetrations, distrib- neighbor, xVM. uted at different planes of the xVB, are A summary of the results obtained in depicted in figure 2. all penetrations is depicted in figure 3, The results obtained showed that the which includes a schematic thalamic body surface is represented with its rostro- “didelphunculus” represented at AP plane

3.7 5

4.75 Fig. 2 The observations made in 34 of the 87 penetrations traversing the xVB are illustrated at three different AP planes. Responses evoked by natural stimulation of the rhinarium (dark areas), head (light stippling), forelimb (clear areas) and trunk, hindlimb and tail (heavy stippling), as shown in the figurine, are indicated in columns representing the various penetrations in the xVB. The surrounding structures are labeled: C, nucleus C; lme, lamina medullaris externa; PF, nucleus parafascicularis; Po, nucleus posterioris thalami; xVL, nuclei ventralis thalami lateralis; xVM, nuclei ventralis thalami medialis. VENTROBASAL COMPLEX OF THE OPOSSUM 237

F face FI forelimb HI hindlimb Ah rhinariurn T trunk t tail Vi vibrissaa

Fig. 3 Semidiagrammatic reconstruction of the ventrobasal complex between the planes A5.25 and A3.75 indicating the areas of representation of the various body regions. Result obtained from the data of 87 penetrations. A thalamic “didelphunculus” is included representing the total body surface as observed in plane A4.75.

4.75. From this reconstruction it is clear region thalamic responses were readily that the body surface is represented at this evoked by tactile stimulation. level as a distorted image reflecting the Similar predominance of deep receptors functional significance of its various parts, in the lumbar and sacral regions was ob- the trigeminal component occupying more served recording first order afferent fibers than 50% of the volume of the nucleus. at the dorsal root in this same species Using natural stimulation the activity (Oswaldo-Cruz et al., ’65). evoked within the xVB was restricted to Neural activity with different character- the contralateral side. Occasionally some istics could occasionally be evoked by light response could be obtained from ipsilateral tapping, by pinching or by applying pres- stimulation in the rhinarium, interior of sure to large areas of the body surface, the mouth, or tip of the lips. Ipsilateral contra- and ipsilateral to the recording projections from the rhinarium in the site. Correlation with the anatomical data opossum were reported by Pubols and indicates that this activity was recorded at Pubols (’66) and by Bombardieri and John- nuclei C, parafascicularis (PF), ventralis son (personal communication). The latter thalami lateralis (xVL), posterioris thal- investigators also found ipsilateral projec- ami (Po) and occasionally at zma incerta tions to xVB from the upper incisors; how- (ZI). ever, all other oral projections were con- The results obtained from recording unit tr a1 a ter a1 . cluster responses to tactile stimulation in A complete coverage of the whole vol- this species are similar to those described ume of the ventrobasal complex was by Pubols and Pubols (’66) in the North achieved through the 87 penetrations, American variety, and comparable to those no silent area being observed upon nat- reported in various eutherian mammals by ural stimulation. Displacement of vibris- other authors. sae, hairs or light tactile stimulation in Based on a single unit analysis and in glabrous regions was effective in evoking neuroanatomical evidences Erickson et al. unit cluster responses, suggesting a high (’64), reported that the ventrobasal com- predominance of this type of somatic sensi- plex of the North American opossum pre- bility. In the caudal half of the body a sented a primitive form of thalamic organi- predominance of deep sensibility was ob- zation in which this complex displayed served, except at the pedimanus. In this properties akin to those found in the pos- 238 A. P. B. SOUSA, E. OSWALDO-CRUZ AND R. GATTASS terior group (Po) of cats as described by Poggio and Mountcastle ('60). 12 7-1 Recording unit cluster activity in a population biased by a high predominance of tactile units would tend to obscure a dabc d minority of units displaying wide receptive fields and polysensory convergence. I \I \/& In order to clarify this matter a second group of experiments was carried out to evaluate the functional properties of single units isolated within the boundaries of the ventrobasal complex. The results of a typical microelectrode e fg hi penetration are shown in figure 4. In this particular penetration 14 units were isolated and studied. Additional information k n regarding multiunit (MU) recording was \ I also included in the original protocol. This penetration traversed the xVB at an AP rn plane similar to that of figure 1, but approximately 0.5 mm medial to the latter. From the MU records and from the receptive fields of the isolated units a gradual shift of the active site becomes clear. At this particular penetration all units isolated displayed rapid adaptation and were driven Fig. 4 Localization and extent of the receptive fields of 14 units isolated along one micro- either by hair displacement or by lightly electrode penetration. On the head region eight touching the skin with a von Frey type units were activated by displacement of vibrissae hair stimulator. or tactile hairs, all of them showing quick adap- The predominance of txigeminal inner- tation. Another unit displayed a restricted field located at the rhinarium. Of the five units isolated vation in this species is reflected in the in the forelimb, three were activated by hair dis- high percentage of units related to the placement in restricted fields on the pre-axial head, representing approximately 65% of surface (units j, 1, n), one m, by light touch on the units isolated, the remaining body sur- the glabrous skin of the tip of digit 11, Unit k responded to deep stimulation. face being represented to a lesser degree (see table 1). In the head region the vibrissae and other tactile hairs correspond to a Receptive fields on the pinna and on the high proportion of the population; the re- hairy surface of the head were elongated, maining receptive fields on hairy regions, being oriented along the anatomical axis. rhinarium and lips displayed round or oval Tactile receptive fields in the glabrous shape, similar in area and in distribution skin of the rhinarium and lips measured to those reported in other species (Darian- only a few square millimeters, being at Smith, '64). Typical receptive fields of the times punctiform. head region are illustrated in figure 5. Of The receptive fields in other body re- the 286 units studied 41% were activated gions cover wider areas being larger at the by mechanical stimulation restricted to a trunk and axial portions of the limbs. EX- single tactile hair; of these some responded amples of such receptive fields are given in to a specific direction of displacement. Of figure 6. The receptive fields of the fore- the tactile hairs present in the head region limb are larger in its axial portion, de- the snout vibrissae displayed the highest creasing in size towards the extremities, innervation density. Units related to the as reported in other eutherian mammals supraorbital, zygomatic and mandibulary (Mountcastle et al., '57; Pubols et al., '65). tactile hairs were also isolated, comprising The receptive fields on the hindlimb OC- 6% of the population studied. cupy a larger area except at the pedimanus VENTROBASAL COMPLEX OF THE OPOSSUM 239

TABLE 1

8 % Head 186 65 Face rhinarium 22 7.7 vibrissae 87 30.4 face 77 26.9 Forelimb 87 30.4 Forelimb hand 69 24.1 arm and forearm 18 6.3

Trunk 7 2.4 Trunk ~7 2.4 Hindliinb 6 2.1 Hindlimb G 2.1

1270 138e mately 1% of the population, would not fit this account. These units were driven by ipsilateral stimulation or displayed discontinuous fields. In a group of nine animals the xVB was explored while the animals were electri- cally stimulated, simultaneously or singly at eight different points (see Material and Methods). Units isolated under these conditions were found to have the same characteristics as those studied under natural stimulation. Under electrical stimulation, units within the ventrobasal complex were activated only when their restricted receptive field extended over one of the stimulation sites. In this series of experiments and in the previous one, no units within the xVB were activated by auditory 134sa 138 h 138, 129aa 129 r stimulation. In order to rule out the depressing action of sodium pentobarbital in multisynaptic pathways, five animals were anesthetised with dial-urethane and two animals with a-chloralose, which is known to enhance multisensory responses (Kruger and Albe- Fessard, ’60). , 135e 125 f 138 vv “Protopathic” projections are conducted Fig. 5 Receptive fields located in the head by the antero-lateral system, the fibers of region of 11 animals, illustrating 32 units acti- this system projecting also to the xVB, as vated by displacement of tactile hairs, by strok- shown by anatomical and electrophysio- ing the fur in restricted areas, or by light touch logical techniques in other species (for a in glabrous skin of the pinna, rhinarium and lips. review see Bowsher, ’65). These responses where they are similar in size to those are possibly controlled or overridden by the found in the hands. Of the 286 isolated lemniscal projections. In order to ascer- units, approximately 80% were driven by tain the nature of these projections in this surface stimulation, either by hair dis- marsupialian ventrobasal complex a series placement or light touch (see table 2). of experiments were carried out with the Stimulation of deep structures activated dorsal columns sectioned at high cervical 16% of the units but, surprisingly, no levels, as suggested by Pubols and Pubols units were found to be driven by joint dis- (’66). placement. In two animals the dorsal column was Of the 286 units isolated within the ana- sectioned bilaterally at a level rostra1 to tomical boundaries of the ventrobasal com- the first rootlet of GI. In the animals plex, only three, representing approxi- multiunit records and ten isn- 4~ 240 A. P. B. SOUSA, E. OSWALDO-CRUZ AND R. GATTASS

------'7 \ IL, . 128 0 128u ------1 134h' ' 1138ff 128f Fig. 6 Localization of the receptive fields of 12 units in the trunk and limbs in seven experiments. There is a gradual decrease in size in the receptive fields towards the extremities of the limbs. At the digits they reach a size as small as those observed in the rhinarium. This reduction in size is more evident in the hindlimb, as comparing the fields of units 125, or 138ii to those of units 138ee and 138ff.

TABLE 2 An anatomical investigation in newborn Skin Deep and in adult specimen of the opossum demonstrated the presence of a lateral cervical hair touch joint deep Other nucleus located at the first cervical seg- 163 72 0 46 3 ment (unpublished results ). The func- 57.4% 25.4% 0% 16.2% 1.1% tional properties of the lateral cervical nucleus in this species is undergoing study displaying receptive fields in the trunk and in this laboratory. limbs, showed properties similar to those In order to rule out the effects of the observed in animals with intact dorsal spino-cervico-thalamic projections two ad- columns, with a predominance of tactile ditional animals were prepared with the units. Although the background activity dorsal column sectioned at Ci and both evoked by natural and electrical stimula- dorsolateral quadrants sectioned rostra1 to tion of the trunk and limbs was less in- Cz. Under these circumstances projections tense than in animals with intact dor- to the xVB were then restricted to the sal columns, "lemniscal" projections still spino-thalamic tract which is rudimentary reached the ventrobasal complex. It is well known that the spino-cervico-thalamic in the opossum (Mehler, '57). tract displays a predominance of the lem- In these animals 19 units located within niscal type of projection (Ha et al., '65; the ventrobasal complex were isolated and Morin et al., '63; Gordon and Jukes, '63; studied. This sample showed a predomi- Oswaldo-Cruz and Kidd, '64; Kitai et al., nance of units responding to stimulation '65; Horrobin, '66) and that the ventro- of deep structures of the limbs and trunk basal complex is the thalamic site of achieved by applying pressure, by tapping termination of this pathway (Morin and or occasionally only by electrical stimula- Catalano, '55; Morin and Thomas, '55; tion. Receptive fields observed in this sam- Busch, '61; Landgren et al., '65; Oswaldo- ple were all contralateral and comparable Cruz, '65; Andersen et al., '66). in area to those isolated in intact animals. VENTROBASAL COMPLEX OF THE OPOSSUM 24 1

None of the above mentioned units re- group in cats has been described by various sponded to auditory stimulation. authors (Diamond and Utley, ’63; Morest, In this series units responding to electri- ’65; Oswaldo-Cruz and Rocha-Miranda, cal stimulation of widely dispersed areas ’67a, ’68). Although no systematic analy- were also observed lying in surrounding sis has been made in this region, the ob- structures. servations of a large number of isolated Responses to somesthetic stimulation units yielded results identical to those were also recorded in nuclei surrounding described in the cat and monkey (Poggio the ventrobasal complex. As the electrode and Mountcastle, ’60; Perl and Whitlock, approached the dorsal border of the xVB ’61; Whitlock and Perl, ’59, ’61). A small neural activity could occasionally be percentage of the units displayed restricted evoked by tapping or applying pressure to peripheral fields, similar in all respects to the ipsi and contralateral body surface. those observed within the ventrobasal com- The region promoting neural discharges plex. Other units displayed wide receptive was not correlated with the body region fields, often discontinuous and frequently represented in the underlying ventrobasal bilateral. A large number of units showed complex. In various penetrations weak ac- sensory convergence being also driven by tivity evoked by intense stimulation of the auditory stimuli. At the level of the caudal forelimb was abruptly substituted by typi- pole of the ventrobasal complex, units iden- cal ventrobasal activity related to the head tified as belonging to nuclei parafascicu- area. This type of activity observed at the laris and subparafasciculnris presented ex- dorsal border of xVB can be seen in the tensive receptive fields under electrical schematic representation of some penetra- stimulation. An example of one of these tions depicted in figure 2. These penetra- units isolated in the parafascicular nucleus tions, represented by columns, interrupted is shown in figure 7. This cell was activated at the dorsal boundary of the ventrobasal by ipsi- and contralateral electrical stimu- complex, show regions activated by intense lation of the forelimbs, rhinarium and forearin stimulation dorsal to the repre- face. sentation of the head region. This exten- sion of atypical activity into the limits of DISCUSSION the ventrobasal complex possibly results According to the evidence presented by from errors in estimating the bottom of Herrick (‘48) and the interpretations of the tract and in applying the corrective fac- Bishop (’59) and Diamond and Chow tors involved in the establishment of the (’62), the thalamus evolves from an un- vertical scale. Another cause of error lies differentiated cell group receiving fibers in the transposition of the various pene- from different sources, as seen in lower trations, performed in different experi- forms, to an aggregate of differentiated nu- ments, to a single “standard plane.” clei, each receiving restricted projections, Activity of this type was found in a as found in higher mammals. According to shell-like region capping the dorsal and these arguments marsupials, being “primi- dorsolateral borders of the xVB including tive” mammals, should display an inter- parts of the ventrolateral complex and nu- mediate form of thalamic organization. cleus C, regions described in detail in pre- Anatomical investigations of the thala- vious publications (Oswaldo-Cruz and mus of the commun opossum have shown Rocha-Miranda, ’67a, ’68). Similar results an organization akin to that displayed in were also obtained by various authors other mammals, and homologies have been under different ewerimental conditions drawn between the nuclear masses found (Kruger and Albe-Eessard, ’60; Oswaldo- in the opossum and those described in Cruz, ’61; Albe-Fessard and Bowsher, ’63; eutherian mammals (Bodian, ’39; Walker, Albe-Fessard and Bowsher, ’65). ’38). These “primitive” mammals which Activity evoked by natural or electrical show a low degree of neo-cortex develop- stimulation of the body surface was also ment (Gray, ’24) present a thalamo-corti- recorded in the region of the posterior cal organization similar in various aspects group. In the opossum a region with char- to that of more developed mammals (Loo, acteristics homologous to the posterior ’30, ’31; Bodian, ’42). The eutherian pat- 242 A. P. B. SOUSA, E. OSWALDO-CRUZ AND R. GATTASS

Fig. 7 Responses of unit 148p isolated in nucleus parafasciculuris, to contralateral (contra) and ipsilateral (ipsi) electrical stimulation of the four limbs, face and rhinarium. (Rh, rhinarium; F, face; F1, forelimb; H1, hindlimb). This unit responds to bilateral stimulation of the rhinarium, face and forelimbs, being silent to stimulation of the hindlimb. Another unit, of smaller amplitude, responds to bilateral stimulation of the hindlimbs. tern of thalamo-cortical organization was displayed by higher mammals. Neverthe- not found by Diamond and Utley ('63) in less certain characteristics, such as corti- the North American opossum. According to cal architectonics and the overlap of motor these authors all thalamo-cortical connec- and somatosensory cortical representation, tions in this subspecies are of thc sustain- suggest a primitive pattern of organization. ing type. Different results may stem from Regarding the functional organization of a spread of the lesion to the white matter, the xVB in the opossum, conflicting results as consequence of the peculiar vascular are found in the literature (Erickson et arrangement in this species (Wislocki and al., '64; Pubols and Pubols, '66). The re- Campbell, '37). sults obtained in the present experimental In spite of the fact that the opossum series lend support to those reported by shows a poorly developed neo-cortex, well- Pubols and Pubols ('66). circumscribed sensory receiving areas have Somatotopic organization. Our record- been shown both in the North and South ing of unit cluster responses in the dorsal American subspecies (Lende, '63a; Rocha- thalamus of the D. marsupialis aurita Miranda and Oswaldo-Cruz, unpublished showed a precise soma totopic organiza- results), as well as in the closely related tion. The body surface displays a distorted marsupial, Didelphis azarae azarae Tem- representation reflecting, in a striking way, mink, 1825 (Saraiva and MagalhHes Cas- the functional significance of each body tro, '69; MagalhHes Castro and Saraiva, area, regions of greater innervation density '69). On the other hand the opossum shows occupying a larger thalamic volume. In a marked departure in the neural organi- this representation the head projects to zation when compared to the higher mam- the medial portion of the nucleus and the malian pattern; somato-sensory and motor tail occupies an anterolateral position. representation overlap at cortical level. This pattern is similar to that described in This overlap of function has been reported the cat (Mountcastle and Henneman, '49) in other marsupials (Adey and Ken, '54; and in the rat (Emmers, '65). A caudo- Lende, '63a ,b ) . lateral representation of the tail region was From the papers discussed above it be- reported by Welker and Johnson ('65) in comes clear that marsupials conform in the raccoon. Our results are substantially most respects to the neural organization in agreement with those obtained by VENTROBASAL COMPLEX OF THE OPOSSUM 243

Pubols and Pubols ('66) except for the Similar observations were made ventral fact that these authors did not find a tail to the xVB in the region occupied by zoria representation, possibly due to the limited incerta and the lateral portion of Campi number of penetrations in the lateral Forelk Activity evoked by somatic stimu- region of the ventrobasal complex. Bom- lation has also been reported in this region bardieri and Johnson (personal communi- in different species under various anes- cation) have also found a tail representa- thetic conditions (Oswaldo-Cruz et al., '56; tion in the xVB of the North American Albe-Fessard et al., '59; Kruger and Albe- opossum. Small discrepancies between our Fessard, '60; Oswaldo-Cruz, '61; Darian- results and Pubols' on the topographical Smith, '64; Denavit et al., '64; Albe-Fessard organization at various AP planes may be and Bowsher, '65). Units isolated within attributed to differences in the adopted zona incerta display wide receptive fields, often bilateral. These regions have been plane of sectioning. found to receive spinal afferents from the Relation to cytoarchitecture. No corre- lateral columns (Nauta and Kuypers, '57; lation could be established between sub- Hand and Liu, '66; see also Herrick, '26). divisions based in the cytoarchitecture of At the posterior border of the ventro- this complex and the projections of the basal complex convergent type of activity various body regions as observed in other was recorded in the posterior group and in species, which present a clear lamination the parafascicular nuclei. A great percen- pattern within this nuclear mass (see tage of units isolated in Po and the ma- Welker and Johnson, '65). jority of those in nuclei parafascicularis Question of diffuse projections. Diffuse and subparafascicularis were activated by projections resulting in convergent re- stimuli noxious in character. Units pre- sponses at a given site of the xVB have senting somato-auditory convergence were been reported by several authors record- observed in the posterior group. ing with macroelectrodes (Cohen and Grundfest, '54; Gaze and Gordon, '54; and CONCLUSIONS others). These results can be attributed to The topographic representation of the the spread of electrical fields in a volume body surface in the ventrobasal complex conductor (see Woodbury, '62). The syn- of the opossum, revealed by gross electrode chronous firing of a large neuronal popu- exploration, was confirmed and supple- lation of the skin or nerve trunks will gen- mented by single unit recording. The func- erate an extensive electrical field. With tional properties of units isolated in this the macroelectrodes employed in the pres- complex are identical to those reported in ent study all responses recorded in the eutherian mammals, i.e., they display ventrobasal complex showed a discrete place- and modality-specificity. A great point to point representation, activity being predominance of skin representation was evoked by somatic stimulation, while visual observed throughout the nucleus, receptive and auditory stimuli were ineffective. fields being comparable in size and orien- Responses from neighboring structures. tation to those reported in carnivores and Small amplitude responses to intense ipsi- primates, both in the trigeminal and in lateral or bilateral somatic stimulation the spinal territories (for a review see showing gross somatotopic organization Mountcastle and Darian-Smith, '68). When were found in nearby structures above the compared to other species, the opossum dorsal border of the ventrobasal complex. shows a smaller percentage of deep projec- These responses are possibly homologous tions, and although special care was taken to those reported in rodents (Libouban- in this respect, no units were found to be Letouze, '64), carnivores (Kruger and activated by joint displacement. Of all Nbe-Fessard, '60) and primates (Oswaldo- units studied, only a small percentage com- Cruz, '61; Albe-Fessard and Bowsher, '65). prising approximately 1% of the popula- This type of evoked activity is greatly en- tion, displayed properties differing from hanced by the use of a-chloralose as anes- the lemniscal type of activity. A large num- thetic agent, being absent or of small ber of units with identical properties were magnitude in animals anesthetised with recorded in nearby structures, and so a barbiturates. possibility arises that these units allocated 244 A. P. B. SOUSA, E. OSWALDO-CRUZ AND R. GATTASS to the ventrobasal complex belong in real- column-medial lemniscus system, single ity to the xVL complex, nucleus C or other unit recording of the ventrobasal complex nearby structures. was made in animals after sectioning of Our mapping experiments using unit the dorsal columns. Under these conditions cluster responses and the observations of projections to the ventrobasal complex multiunit records and the receptive fields were restricted to the neo- and paleospino- of isolated single units are in close agree- thalamic tracts. Recording in animals with ment to the results obtained by Pubols and lesions restricted to the dorsal columns Pubols ('66 j in D. virginiana being in total demonstrated that although there was a variance to the results reported by Erick- marked reduction in the number of elements activated by natural stimulation of son et al. ('64). The discrepancies ob- the body surface below the level of sec- served could stem from four sources: tioning, the great majority of units re- ( 1j sampling technique; microelectrodes corded displayed lemniscal properties. of different type, displaying different re- Additional lesions in the dorsolateral quad- cording properties, could introduce a bias rant, contralateral to the recording site, by rejecting part of the neuronal popula- suggested a projection similar to the spino- tion, i.e., "smaller neurons"; (2) stimula- cervico-thalamic tract as described in tion technique; sampling of a population carnivores (for a review on the subject driven by natural stimulation could place see Bowsher, '65 j. The results obtained in a special emphasis on those units readily animals with the thalamic input restricted activated by natural stimuli, leaving un- to the ipsilateral anterolateral quadrant detected those displaying wide receptive were similar to those described by Whit- fields or those activated by more intense lock and Per1 ('59 j in the cat. Under these stimulation; (3j preferential activation of conditions there was a considerable reduc- the dorsal column-medial lemniscus sys- tion in the number of active elements that tem caused by natural stimulation would can be justified by the poor development override and obscure the effect of sensory of the spino-thalamic tract in the opossum, information conducted by small diameter as shown by Mehler ('57). fiber spectrum; (4) sodium pentobarbital The results obtained in the various types and other barbiturates tend to block pref- of experiments indicated that the ventro- erentially some types of response and have basal complex of the South American also been shown to reduce and modify re- opossum, D. marsupialis aurita, displays a ceptive fields of cells in the posterior group functional organization similar to that (Poggio and Mountcastle, '60). described in eutherian mammals. Our re- In a series of experiments we tried to sults confirm and supplement the topo- duplicate the conditions in which Erickson graphical organization reported by Pubols et al. ('64) reported a high percentage of and Pubols ('66) in the North American wide fields units in the ventrobasal com- subspecies. The results of the single unit plex in the opossum. Single unit activity analysis are in complete variance to those was recorded in opossums anesthetised presented by Erickson et al. ('64) in the with Dial urethane in response to electri- opossum, as well as with those reported by cal stimulation applied simultaneously or the same group in the posterior dorsal singly to different points of the body sur- thalamus of the hedgehog (Erickson et al., face. The results obtained were identical '67). The observed discrepancies cannot to those observed under natural stimula- be attributed to differences in technical tion in animals anesthetised with sodium procedures involved in stimulation and re- pentobarbital. Resorting to a-chloralose, an cording methods. Single units displaying anesthetic known to enhance polysensory properties similar to those reported by responses, yielded the same negative re- Erickson et al. ('64) in the ventrobasal sults. Summarizing, using different re- complex were observed in structures sur- cording and stimulating techniques and rounding this nuclear mass. different anesthetic conditions (Nembutal, Dial-urethane and x-chloralose) identical ACKNOWLEDGMENTS results were obtained. In order to ascertain The authors wish to express their grati- the possible role exerted by the dorsal tude to Dr. C. E. Rocha-Miranda and Mr. VENTROBASAL COMPLEX OF THE OPOSSUM 245

R. Lent for their participation in some of Bowsher, D. 1965 The anatomophysiological the experiments; to our technician Mr. basis of somatosensory discrimination. Intern. K. Rev. of Neurobiology, 8: 35-75. F. Bernardes for his unfailing and skillful Busch, H. F. M. 1961 An anatomical analysis assistance in all phases of this work. We of the white matter in the brain of the cat. also express our gratitude to Prof. J. I. Thesis, VanGorcum & Co., N.V., 116 pp. Cabral, R. J., and J. I. Johnson 1971 The or- Johnson for the valuable advices and criti- ganization of mechanoreceptive projections in cal appraisal of the manuscript. the ventrobasal thalamus of sheep. J. Comp. Neur., 141: 17-36. LITERATURE CITED Carreras, M., and S. A. Andersson 1963 Func- tional properties of neurones of the anterior Adey, W. R., and D. I. B. Ken 1954 The cere- ectosylvian gyrus of the cat. J. Neurophysiol., bral representation of deep somatic sensibility 100-126. the marsupial phalanger and rabbit. 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