thalamocortical and intracortical projections to the forelimb-stump si representation of rats that...

Thalamocortical and IntracorticalProjections to the Forelimb-Stump SI

Representation of Rats That SustainedNeonatal Forelimb Removal

ANDREY S. STOJIC,1 RICHARD D. LANE,1* HERBERT P. KILLACKEY,2

BABAR A. QADRI,1 AND ROBERT W. RHOADES1

1Department of Anatomy and Neurobiology, Medical College of Ohio, Toledo, Ohio 436992Department of Psychobiology, University of California, Irvine, Irvine, California 92717

ABSTRACTWe previously reported the abnormal expression of hindlimb receptive fields in the stump

representation of the primary somatosensory cortex (SI) in rats that sustained neonatalforelimb removal when cortical g-aminobutyric acid (GABA) receptors were pharmacologi-cally blocked (Lane et al. [1997] J. Neurophysiol. 77:2723–2735). In this study, we attemptedto identify the substrate for this functional modification. Three potential substrates wereexamined: 1) changes in intracortical connections within SI; 2) alterations in the projectionpattern of thalamocortical afferents from the ventroposterior lateral (VPL) nucleus to SI; and3) changes in the receptive fields of thalamocortical neurons. We used biotinylated dextranamine and Phaseolus vulgaris leucoagglutinin to examine the intracortical projectionsassociated with the stump and hindlimb representations of SI. True Blue and DiamidinoYellow were used to study the organization of the VPL projections to SI. Finally, single-unitrecordings from VPL neurons were made to examine the functional organization of thisnucleus in neonatally amputated adult rats. Tracer studies demonstrated no significantchange in the intracortical connections or VPL projections associated with the stump andhindlimb SI in neonatally amputated rats. Recordings from VPL of neonatally manipulatedrats revealed a small, but significant, population of cells (19.0%) within the stump representa-tion that had dual stump and hindlimb receptive fields. Thus, the data suggest that thefunctional reorganization observed in SI of neonatally amputated rats may reflect functionalalterations occurring in its thalamic inputs. J. Comp. Neurol. 401:187–204, 1998.r 1998 Wiley-Liss, Inc.

Indexing terms: biotinylated dextran amine; Phaseolus vulgaris leucoagglutinin; rodent;

somatosensory and ventroposterior lateral nucleus

Recent experiments from this laboratory indicated thatneonatal forelimb removal in rats results in invasion of thecuneate nucleus (CN) by sciatic nerve afferents and thedevelopment of cuneothalamic neurons with receptivefields that include both the hindlimb and forelimb-stump(Lane et al., 1995). This alteration in subcortical organiza-tion is associated with a change in cortical somatotopy thatcould be revealed when g-aminobutyric acid (GABA) recep-tors were blocked in cortex; numerous sites in the corticalstump representation became responsive to hindlimbstimulation (Lane et al., 1997). This finding raised severalquestions, including the following: what is the substratefor the functional change observed during cortical GABAblockade? There are several potential substrates for theobserved changes.

First, the cortical change may reflect alterations in thefunctional organization of subcortical regions of the somato-sensory system, namely, the sensory thalamus. Evidencefrom experiments in rats (Verlay and Onnen, 1981; Rhoadeset al., 1987; Nicolelis et al., 1991, 1993; Chiaia et al., 1992;Alloway and Aaron, 1996), raccoon (Rasmusson, 1996),monkey (Garraghty and Kaas, 1991), and humans (Kiss et

Grant sponsor: National Institutes of Health; Grant numbers: NS 28888and DE 07734.

*Correspondence to: Richard D. Lane, Department of Anatomy andNeurobiology, Medical College of Ohio, Toledo, OH 43699.E-mail: [email protected]

Received 14 August 1997; Revised 8 June 1998; Accepted 23 June 1998

THE JOURNAL OF COMPARATIVE NEUROLOGY 401:187–204 (1998)

r 1998 WILEY-LISS, INC.

al., 1994) suggests that injury-induced functional reorgani-zation does occur in the thalamus and thus could explainthe alterations seen in sensory cortex following peripheralnerve injury. In a recent report that examined the mecha-nisms involved in large-scale cortical reorganization inadult monkeys with hand and forearm amputations, Flor-ence and Kaas (1995) suggested that changes in subcorti-cal regions of the sensory neuraxis could in large part, ifnot totally, serve as the substrate for functional plasticityobserved in the somatosensory cortex.

Another possible explanation for the functional reorgani-zation seen in primary somatosensory cortex (SI) of ratsthat sustained neonatal forelimb removal may be changesin projections from the ventroposterior lateral (VPL)nucleus to SI. Few studies have explored this issue, butVerney et al. (1982) provided evidence for reorganization ofthalamocortical projections from the ventrobasal complex(VB) of mice that had their mystacial vibrissae removed atbirth. Conversely, McKinley and Kruger (1988) reportedno anatomical changes in the projections of VPL to somato-sensory cortex in adult cats following T12 spinal cordtransection at 2 weeks of age.

Finally, changes in the intracortical connections of SIitself could account for the functional reorganization seenat this level. This mechanism could involve the strengthen-ing of latent, preexisting intracortical connections betweenthe hindlimb and forelimb SI representations followingneonatal forelimb removal. Such a possibility has beendiscussed in previous reports (Wall, 1988; Jenkins et al.,1990; Smits et al., 1991; Hirsch and Gilbert, 1993; Li andWaters, 1996; Li et al., 1996). On the other hand, acorticocortical pathway could emerge de novo followingperipheral deafferentation via collateral sprouting. Suchanatomical reorganization has been reported by Darian-Smith and Gilbert (1994), who observed sprouting ofintracortical axons into regions of cat visual cortex thathad been deprived of visual inputs.

In this study, we examined the consequences of neonatalforelimb removal on the organization of intracortical andthalamocortical fiber projections to the forelimb-stumprepresentation. In addition, the functional organization ofVPL was examined via electrophysiologic recordings to

determine the degree to which CN reorganization wasexpressed at the thalamic level.

MATERIALS AND METHODS

All protocols described here were developed in accor-dance with the National Institutes of Health Guide for theUse of Laboratory Animals and were approved by theInstitutional Animal Care and Use Committee of theMedical College of Ohio.

Neonatal forelimb removal

Neonatal forelimb removals were carried out as previ-ously described (Lane et al., 1995, 1997). Briefly, P0 rats(.12 hours old) were anesthetized by hypothermia, andthe left forelimb was amputated just distal to the shoulder.The skin was closed with cyanoacrylate following cauteriza-tion of the brachial artery and application of 0.7% bupiva-caine. The pups were then warmed and returned to theirmothers.

Anterograde and retrograde labeling ofintracortical projections to the forelimb

region of SI with biotinylated dextran amine

Ten adult rats (.60 days old, four normals and sixanimals that sustained neonatal forelimb removal) wereused in this experiment. Rats were anesthetized withketamine (60 mg/kg) and xylazine (15 mg/kg) adminis-tered intraperitoneally. They were placed in a stereotaxicheadholder and a midline incision was made to expose theskull. A portion of the skull was removed, revealing asmall area of the right cerebral cortex corresponding to theforelimb representation of SI (typical coordinates of ex-posed cortex were 1.0 mm anterior and 4.0 mm lateral withrespect to bregma [Paxinos and Watson, 1982]). Confirma-tion of the location was made by multi-unit recording andreceptive field mapping. Micropipettes (tip diameter 70–100 µm), filled with a 10% biotinylated dextran amine(BDA) solution (Molecular Probes, Eugene, OR) in 0.1 Msodium phosphate-buffered saline (PBS), pH 7.4, werelowered to a depth between 600 and 800 µm below thecortical surface (approximate location of cortical laminaIV), and BDA was ejected ionophoretically by applying a5-µA positive current for 1.5 hours. At the end of this time,the electrode was removed and the wound closed.

Seven to 8 days following the procedure, two normal ratsand four neonatally manipulated rats were euthanizedwith carbon dioxide and perfused, initially with heparin-ized PBS, immediately followed by a 4% paraformaldehydesolution. The right cortex of each brain was removed,flattened, and cut into 50- µm-thick sections on a freezingmicrotome. Alternate sections were stained for BDA (Hoef-linger et al., 1995). The remaining sections were counter-stained for cytochrome oxidase (CO) using the method ofWong-Riley (1979).

For the remaining rats (two normal rats and two ratsthat sustained neonatal forelimb removal), the followingrecording experiment was performed before the animalswere euthanized. Seven to 8 days following BDA injection,rats were anesthetized and prepared for recording aspreviously described (Rhoades et al., 1993; Lane et al.,1995). The animals were then placed in a stereotaxicheadholder. A midline incision was made to expose theskull and posterior aspect of the neck. Muscles of theposterior neck were then reflected to reveal the medulla.

Abbreviations

AS anterior snoutBDA biotinylated dextran amineCN cuneate nucleusCO cytochrome oxidaseDC dysgranular cortexdLGN dorsal lateral geniculate nucleusDY diamidino yellowFL forelimbGABA g-aminobutyric acidHL hindlimbLJ lower jawnRT nucleus reticularis thalamiPBS phosphate-buffered salinePHA-L Phaseolus vulgaris leucoagglutininRF receptive fieldSI primary somatosensory cortexST StumpT trunkTB True BlueV vibrissae (mystacial)VPL ventroposterior lateral nucleusVPM ventroposterior medial nucleusVPo ventroposterior nucleus

188 A.S. STOJIC ET AL.

The dura and arachnoid were cut at the base of the skull toallow for drainage of cerebral spinal fluid. A craniotomywas performed over the right cerebral cortex, contralateralto the amputated forelimb, which exposed most of thecerebral cortex. The dura and arachnoid were reflectedand warm silicone fluid was used to cover the brain. Adrawing of the cortex was made at 443 magnification torecord the placement of microelectrode penetrations.

Mapping was performed using procedures described inearlier studies (Rhoades et al., 1993; Lane et al., 1995).Briefly, unit clusters were recorded with varnish-coatedtungsten electrodes (Z 5 0.9–1.3 MV). Cutaneous recep-tive fields were defined by tactile stimuli delivered withblunt probes. Electrode penetrations were spaced approxi-mately 200 µm apart and multi-unit activity was recordedat depths between 500 and 750 µm. Between 20 and 50penetrations were made, to map the functional borders ofthe forelimb-stump and hindlimb SI representations. Oncethe functional borders of these two areas were defined, therecording microelectrode was replaced with a second tung-sten microelectrode that was coated with a 2.5% True Blue(TB) solution (Sigma, St. Louis, MO) dissolved in distilledwater. The electrode was left in the cortex for approxi-mately 2 minutes to allow dye uptake by the surroundingtissue, and then removed. This procedure was repeated sixto eight times per animal (the electrode was recoated withTB prior to each penetration). Approximately three to fourpenetrations were made along the forelimb-stump andhindlimb border and three to four penetrations along thelateral border of the forelimb-stump representation. TheseTB injections were made for two reasons. First, theyallowed identification of the forelimb-stump and hindlimbborder, which facilitated analysis of BDA labeling asviewed in coronal sections of the cortex. Second, the TBinjections permitted functional identification of the bor-ders of the forelimb-stump representation and confirma-tion that the site of BDA injection was in fact within thisregion.

When the recording experiment was completed, theanimals were euthanized and perfused as above. Thebrains were removed and cut into 50-µm-thick coronalsections on a freezing microtome. Alternate sections wereprocessed for BDA labeling as described earlier. Theremaining sections were stained with cresyl violet.

BDA injection into the hindlimbrepresentation of SI

Ten adult rats (four normal rats and six neonatallyamputated rats) were used for this part of the study.Procedures for injecting BDA into SI hindlimb representa-tion were identical to those described above, except the siteof injection was centered in the hindlimb representation ofSI (coordinates 0.5 mm posterior and 2.5 mm lateral withrespect to bregma [Paxinos and Watson, 1982]). Identifica-tion of hindlimb SI at the injection site was confirmed bymulti-unit recording and receptive field mapping.

Cortices from two normal rat cases and four neonatallymanipulated rats were flattened before sectioning andprocessing. The remaining rats (two normal rats and tworats that sustained neonatal forelimb removal) underwentthe same recording and tissue processing described imme-diately above.

Anterograde labeling of SI hindlimbintracortical projections with Phaseolus

vulgaris leucoagglutinin

Four adult rats were used in this experiment (onenormal and three rats that sustained neonatal forelimbremoval). The cortical hindlimb representation of SI wasexposed, pipettes filled with a 2.5% Phaseolus vulgarisleucoagglutinin (PHA-L; Vector, Burlingame, CA) solution(in PBS) were lowered to a depth of 600–800 µm below thecortical surface, and PHA-L was deposited ionophoreti-cally by application of a 5-µA positive current for 45minutes to 1 hour. When the injection was completed, theelectrode was removed and the wound was closed. Animalssurvived between 10 and 14 days; they were euthanizedand perfused as described above. The right cortex of eachbrain was removed and cut as described above, and thenalternate sections were processed for demonstration ofPHA-L (Hoeflinger et al.,1995). The remaining sectionswere processed for CO.

Analysis of BDA- and PHA-L-labeledintracortical cells and fibers

BDA- and PHA-L-stained sections were analyzed in anidentical manner. CO-stained alternate sections were usedto draw a representation of SI. Following this, sectionsstained for either BDA or PHA-L were superimposed overCO-based drawings. Because CO staining results in greatershrinkage of sections than the other staining methods,adjustments in magnification were made to compensatefor the difference in size between the CO-stained and BDA-or PHA-L-stained sections. The positions of cells and fiberslabeled with the tracers in all cortical laminae were thenmarked on the CO map.

In the cases in which brains were cut into coronalsections, analysis was performed to examine the followingissues. First, the BDA-stained sections were viewed underlight microscopy to identify areas of BDA fiber labeling.The same sections were then analyzed with a NikonOptiphot microscope equipped with episcopic fluorescenceoptics. The TB injections used to mark the forelimb-stumpand hindlimb border were viewed with an UV-2A (excita-tion wavelength 360 nm) filter cube. This permitted identi-fication of fibers located in the hindlimb representation(BDA labeling medial to the TB border mark) and fiberslabeled in the forelimb-stump representation (BDA label-ing lateral to the TB border mark). Finally, the BDA-stained sections were compared with the alternate sec-tions that were stained for cresyl violet to evaluate thelaminar distributions of labeled fibers and cells.

Retrograde labeling of thalamocortical cellswith TB and diamidino yellow

Eight adult rats (four normals and four rats that sus-tained neonatal forelimb removal) were used for thisexperiment. The animals were anesthetized as above andplaced in a stereotaxic headholder, and then two smallopenings were made in the skull over the right cerebralcortex: one over the SI forelimb representation and an-other over the SI hindlimb representation. The positions ofthese two representations were confirmed by multi-unitelectrophysiologic recordings. A micropipette (tip diameter, 100 µm) filled with 2.5% TB dissolved in distilled waterwas lowered into the hindlimb representation to a depth of1.0 mm, and 1–2 µl of tracer were pressure injected over a

PROJECTIONS TO FL-STUMP SI 189

period of 10 minutes. Following this, a second micropipettefilled with 2.0% Diamidino Yellow (DY; Sigma) dissolved insaline was placed in the forelimb-stump representation,and 1–2 µl of tracer were then pressure injected as above.Injection micropipettes were removed, and the wound wasclosed.

After 2 days, animals were euthanized with carbondioxide and perfused as above. The right cortex of eachbrain was removed, flattened, and cut into 50-µm-thicksections on a freezing microtome. The thalamus of eachbrain was cut into 50-µm coronal slices on a freezingmicrotome. Alternate cortical sections were plated ontoglass slides, allowed to dry overnight, dehydrated, cleaned,and coverslipped. All thalamic sections were plated andcoverslipped. Alternate cortical sections were processedfor CO.

The sections were analyzed with a Nikon Optiphotmicroscope equipped with episcopic fluorescence optics.TB- and DY-labeled cells were viewed together with aUV-2A (excitation wavelength 360 nm) dichroic filter cube,and DY-labeled cells alone were visualized with a UV-2B(excitation wavelength 460 nm) dichroic filter cube.

Segregation of VPL neurons projecting to the forelimband hindlimb representations within the SI was quantita-tively evaluated in the following way. A circle was drawnaround the population of cells labeled by the hindlimb SIinjection (TB). Cells within this closed contour that werelabeled with DY (injected into forelimb-stump SI) werethen counted. An example of this is shown in Figure 8.

This cell counting was performed on all VPL sections ineach animal. A similar method has been used by Rasmus-son and Nance (1986).

Single-unit recordings from VPL neurons

Seven normal rats and ten rats that sustained neonatalforelimb removal were used for this experiment. Rats wereinitially anesthetized with ketamine (60 mg/kg) and xyla-zine (15 mg/kg) administered intraperitoneally and pre-pared for recordings as described in earlier studies(Rhoades et al., 1993; Lane et al., 1995). The trachea wascannulated, the brachial plexus and sciatic nerves wereexposed, and animals were placed in a stereotaxic head-holder and ventilated mechanically. A bipolar brachialplexus stimulating electrode was placed just proximal tothe origins of the median, ulnar, and radial nerves. An-other stimulating electrode was placed around the sciaticnerve approximately 15 mm distal to the sciatic notch.Rats were paralyzed with gallamine triethiodide (30 mg,delivered intraperitoneally), and light anesthesia wasmaintained during the course of the recording session byadministration of urethane (200 mg, intraperitoneally) asneeded. The cisterna magna was opened and a largecraniotomy was performed over the right cerebral cortex,contralateral to the amputated forelimb. The dura andarachnoid were reflected, and warm silicone fluid was usedto cover the brain.

Single units were recorded with micropipettes (2–20MV) filled with 1 M KCl. Micropipettes were inserted intoregions of cerebral cortex overlying the VPL nucleus usingthe following stereotaxic coordinates: 22.3 mm to 24.3mm rostral-caudally, and 2 mm to 4 mm mediolaterally(Paxinos and Watson, 1982). The recording electrodes werefirst positioned along the rostral-caudal extent of thecortical surface. Following this, the electrode was insertedinto the cortex beginning at 2.0 mm lateral. Subsequent to

completion of recordings from this first track, the electrodewas moved in 0.2 mm increments laterally at the samerostral-caudal position until a final recording track wasmade at 4.0 mm lateral. On occasion, a penetration wouldbe made as far lateral as 4.2–4.5 mm to ensure that thelateral extent of the VPL had been reached. After comple-tion of one such plane of recordings, the micropipette wasthen moved to a new position along the rostral-caudalcoordinates, and the medial to lateral recording schemewas repeated. Typical depths at which electrophysiologicrecordings were monitored and noted were between 4.5mm and 7.0 mm below the cortical surface. Cutaneousreceptive fields were determined by tactile stimulationusing a blunt probe and plotted on body surface drawings.The response latency of each neuron was measured follow-ing electrical stimulation of brachial plexus, sciatic nerve,and SI cortex (single pulses, 0.1 ms duration, ranging from16 to 48 mA). Identification of VPL cells that projected toSI cortex was made by secure responses to 100 Hz electri-cal stimulation and by collision between evoked ortho-dromic and antidromic spikes (Fuller and Schlag, 1976;Pinault and Pumain, 1989).

Because the micropipettes used in these experimentswere of a relatively low impedance, it was possible todetect audibly multi-unit responses from neurons in thearea from where recordings were made. This informationwas used to construct somatotopic maps of VPL. All of theVPL units that were characterized were located within theforelimb representation of the VPL nucleus.

Positions of neurons recorded within the VPL nucleuswere identified by marking several electrode tracks in eachexperiment with electrolytic lesions made with metalelectrodes at locations of units recorded by micropipettes.At the end of each recording session, animals were eutha-nized and perfused by the methods described above. Thebrain was removed and the thalamus was sectioned in thecoronal plane into 50-µm-thick sections and stained withcresyl violet.

Digital processing of figures

All the figures presented in this study were assembleddigitally. In brief, photomicrograph negatives were digi-tized by using a Nikon LS-3510AF 35 mm film scanner.Drawings (Figs. 1, 2, 4–7, 10) were digitized with an EpsonExpression 636 scanner. The digitized images were thenarranged into figures by using Adobe Photoshop for Macin-tosh. The images were adjusted for brightness, contrast,and balance before final printing.

RESULTS

Anterograde and retrograde tracing of SIforelimb representation using BDA

The labeling obtained with a large BDA injection intothe SI forelimb-stump representation of a rat that sus-tained neonatal forelimb removal is shown in Figure 1.Results from the remaining manipulated and normalanimals are charted in Figure 2.

The results from all the animals were very similar. BDAinjection resulted in dense labeling within the forelimb-stump representation (Fig. 1B,D) and sparse labeling ofcells and fibers in regions characterized as dysgranular


Fig. 1. Biotinylated dextran amine (BDA) labeling of intracorticalconnections associated with the stump representation of primarysomatosensory cortex (SI) from a rat that sustained neonatal forelimbremoval. A: CO-stained section through SI cortex. B: BDA injectionsite in a section adjacent to that shown in A. C: Composite drawingdemonstrating the labeled intracortical fibers and cells from allcortical laminae in relation to the SI stump representation. The grayshading represents the area of BDA fiber labeling and the filled blackcircles indicate stained cell bodies. The individual black lines identifystained fibers found outside of the main area of BDA fiber labeling

shown by the gray shading. D–F: High-power photomicrographs takenof the areas outlined by the squares in B. These micrographs showBDA-labeled cells and fibers in SI stump (D), dysgranular cortex (E),and SI hindlimb (F), respectively. The arrows in F show the twolabeled fibers identified in the SI hindlimb area. The locations fromwhich these photomicrographs were taken are indicated by squareslabeled D, E, and F in B. For orientation, the anterior (a) and lateral (l)directions are indicated by the arrows in the top right hand corners ofA–C. Scale bars 5 1 mm for A,B, 10 µm for D–F.

Fig. 2. Comparison of intracortical connections in all corticallaminae associated with the forelimb-stump representation of SIcortices from normal and neonatally amputated rats. A–D: Compositedrawings of four cases in which neonatally amputated rats wereinjected with BDA centered in layer IV of SI stump region. E,F: Twocases in normal rats that were injected in the corresponding SIforelimb area. In each of these representations, the gray shading

indicates the extent of BDA fiber labeling and the black circlesrepresent stained cell bodies. The individual black lines identifystained fibers found outside of the main area of BDA fiber labelingshown by the gray shading. For orientation, the anterior (a) andlateral (l) directions are indicated by the arrows in the top right handcorner of A. Scale bar 5 1 mm.

cortex (Chapin et al., 1987) or intercalated zone (Fabri andBurton, 1991) (Fig. 1E).

Only a few labeled fibers and cells were seen in thehindlimb representation of neonatally amputated rats(e.g., Figs. 1F, 2A,B,D), and they were located along theborder closest to the forelimb or stump representations.Overall, two neonatally amputated rats had two labeledcells each in the hindlimb cortex. No such cells wereobserved in either of the two remaining amputated ani-mals or any of the normal controls.

Evaluation of sections from flattened cortices and frombrain sections in the coronal plane (Fig. 3) showed that theBDA injections involved all cortical laminae. The densestlabeling was in layers I–IV, with lighter staining evident inlaminae V and VI (Fig. 3D). No BDA-labeled cells andfibers were seen within the hindlimb SI region of cortex ofeither two normal or two neonatally amputated animalswhose brains were sectioned in the coronal plane (e.g., Fig.3G–I).

Anterograde and retrograde tracing from SIhindlimb representation using BDA

Injection of BDA into the hindlimb representation re-sulted in only very sparse labeling in the forelimb-stumpfield. Figure 4 shows results from injecting BDA into SIhindlimb representation of a neonatally amputated rat.Most labeled cells and fibers were found within the SIhindlimb area near the injection site (Fig. 4B,D). Cells andfibers were also visible in dysgranular cortex (Fig. 4E) andonly a few labeled fibers were present in the SI forelimb-stump representation (Fig. 4F). The results from this ratare consistent with those from the two normal animals andthe three other neonatally manipulated animals thatreceived BDA injections into the hindlimb representation(Fig. 5). Analysis of BDA labeling in the cases sectioned inthe coronal plane was also similar to those illustrated.BDA-labeled fibers (two) were observed in the stumprepresentation of one neonatally amputated rat. No suchfibers were seen in any other animal.

Anterograde tracing of SI hindlimb regionusing PHA-L

Anterograde tracing from the hindlimb representationwith PHA-L revealed projections (Figs. 6C, 7) very similarto those seen with BDA. Fibers labeled with PHA-L weremainly located within the SI hindlimb representation (Fig.6B,D), with a few scattered fibers labeled outside of thisregion (Fig. 6E). Only a few labeled fibers were observed inthe forelimb-stump representation of one neonatally ampu-tated rat (Figs. 6F, 7).

TB and DY labeling of VPL neuronsprojecting to SI hindlimb and forelimb-stump

representation of SI

Neonatal forelimb removal did not alter the organiza-tion of the VPL projections to the hindlimb and forelimb-stump regions in SI. In all four cases from the neonatallymanipulated rats and the four normal rats, forelimb-stump SI projecting VPL neurons (DY-labeled) and hind-limb SI projecting thalamocortical neurons (TB-labeled)exist as two relatively segregated populations, overlappingto a small extent where the two populations of cells are inclose proximity (Fig. 8).

We observed no double-labeled neurons or large-scalechanges in the projections from VPL in neonatally manipu-lated rats (Fig. 8). The quantitative analysis (Fig. 9)confirmed the lack of significant difference in the organiza-tion of VPL projections to cortex in the normal andneonatally amputated animals. The mean number of cellswithin the hindlimb SI projecting population that were DYlabeled, and thus forelimb SI projecting, was 111 6 27(standard error). In normal rats, the mean number was117 6 26 (P . 0.05).

Functional reorganization in VPL

The results from the electrophysiologic recording experi-ments are summarized in Table 1. In all, 54 neurons wereisolated from the forelimb representation in VPL in sevennormal rats. All the units responded to forelimb tactilestimulation and had an average response latency to bra-chial plexus stimulation of 7.1 6 3.0 ms. None of the cellsresponded to tactile stimulation of any other region of thehead, trunk, hindlimb, or tail or to electrical stimulation ofthe sciatic nerve. Twenty-one of the normal cells studiedwere tested by antidromic stimulation to determinewhether they projected to forelimb SI. Eight of these cells(38.0%) were antidromically activated.

Sixty-three neurons from the forelimb representation inVPL were isolated from ten rats that sustained neonatalforelimb removal. All these responded to tactile stimula-tion of the stump. Fifty-one neurons (81.0%) had singlereceptive fields on the stump and responded to brachialplexus stimulation with an average latency of 11.6 6 4.1ms (P , 0.0001 versus normal rats). Fifty-two percent (n 524 of 46 tested) were antidromically activated from theforelimb-stump representation in SI.

Nineteen percent (12 of 63) of the units recorded fromthe neonatally amputated rats had dual stump and hind-limb receptive fields (e.g., Fig. 10B; P , 0.05 vs. normalrats). Of these, 33.3% (n 5 4) projected to the forelimb-stump SI region as determined by antidromic stimulation(Fig. 10E). The responses of these dual field neurons totactile stimulation of the stump (Fig. 10H1) and hindlimb(Fig. 10H2) were confirmed by stimulation of the brachialplexus (Fig. 10C) and the sciatic nerve (Fig. 10D). Theaverage response latency to brachial plexus stimulation inneurons with dual stump-hindlimb receptive fields was14.4 6 6.5 ms (P , 0.0001 vs. normal rats). For sciaticnerve stimulation, the average response latency of thedual receptive field units was 24 6 8.7 ms.

DISCUSSION

The data reported in the preceding sections supportthree conclusions. First, neonatal forelimb amputationdoes not result in the development of novel intracorticalprojections from the hindlimb to forelimb-stump represen-tation in SI. Second, neonatal forelimb amputation doesnot significantly alter the organization of the VPL projec-tions to SI. Third, single-unit recordings from VPL ofneonatally amputated rats showed a small, but signifi-cantly increased number of VPL neurons with dual stumpand hindlimb receptive fields that project to the stumprepresentation of SI. Taken together, these results sug-gest that the functional reorganization observed in the


Fig. 3. Coronal section of SI cortex showing extent of BDA labelingfrom a rat that sustained neonatal forelimb removal. A: Montagephotomicrograph showing the extent of BDA labeling following injec-tion of BDA in the SI forelimb-stump representation of a neonatallymanipulated rat. The injection was centered in layer IV of SI stumpregion. B: Montage of the same section in A as viewed underfluorescence microscopy with a UV-2A filter cube showing the TBmarks that were used to delineate the SI forelimb-stump representa-tion. The medial mark identifies the location of the physiologic borderbetween the SI forelimb-stump and hindlimb representations.C: Montage of a section adjacent to the one shown in A stained forcresyl violet. This section was used to identify cortical laminae. Thehorizontal arrows in A–C identifiy the same blood vessel as seen in allthree photomicrographs. D: Montage of the section shown in A at ahigher magnification. The horizontal black lines and Roman numerals(I–VI) at the left edge of the montage delineate the cortical laminae.The vertical dashed line in the center of the montage identifies the

location of the physiologic border between the hindlimb and SI stumpareas. The squares identify areas of the SI stump region (E) and SIhindlimb region (G–I) shown in higher magnification in E. E: Higherpower photomicrograph showing BDA labeling of cells and fiberswithin laminae II/III of the SI stump region. F: High-power photomi-crograph of an area of dysgranular cortex (outside of the region shownin D) located lateral to the SI stump representation showing BDAlabeling of cells. G: High-power photomicrograph from laminae Iwithin the SI hindlimb region of the section shown in D. H: High-powermontage photomicrograph from laminae II and III within the SIhindlimb region of the section shown in D. I: High-power photomicro-graph from laminae V within the SI hindlimb region of the sectionshown in D. Note the absence of fiber and cell labeling in this case. Fororientation, the superior (s) and medial (m) directions are indicated bythe arrows in the lower right corner of A. Scale bars 5 2 mm in C(applies to A–C), 500 µm in D, 150 µm in I (applies to E–I).


Fig. 4. BDA labeling of intracortical connections associated withthe SI hindlimb representation from a rat that sustained neonatalforelimb removal. A: CO-stained section through SI cortex. B: BDAinjection site in a section adjacent to that shown in A. C: Compositedrawing demonstrating the labeled intracortical fibers and cells fromall cortical laminae in relation to the SI hindlimb representation. Thegray shading represents the area of BDA fiber labeling and the blackcircles indicate stained cell bodies. The individual black lines identifystained fibers found outside of the main area of BDA fiber labeling

shown by the gray shading. D–F: High-power photomicrographsshowing BDA-labeled cells and fibers in the SI hindlimb (D), dysgranu-lar cortex (E), and SI stump (F), respectively. The arrows in F showthree labeled fibers identified in the SI stump area. The locations fromwhich these photomicrographs were taken are indicated by thesquares labeled D, E, and F in B. For orientation, the anterior (a) andlateral (l) directions are indicated by the arrows in the top right handcorners of A–C. Scale bar 5 1 mm for A,B, 10 µm for D–F.

Fig. 5. Comparison of intracortical connections in all corticallaminae associated with the SI hindlimb representation of corticesfrom neonatally amputated and normal rats. A–D: Composite draw-ings of four cases in which neonatally amputated rats were injectedwith BDA centered in layer IV of the SI hindlimb area. E,F: Twonormal cases that were also injected in the SI hindlimb area. As inFigure 4C (shown again here in D), the gray shading indicates the

extent of BDA fiber staining and the black circles represent stainedcell bodies. The individual black lines identify stained fibers foundoutside the main area of BDA fiber labeling shown by the grayshading. For orientation, the anterior (a) and lateral (l) directions areindicated by the arrows in the top right hand corner of A. Scale bar 51 mm.


Fig. 6. Phaseolus vulgaris leucoagglutinin (PHA-L) labeling ofintracortical connections in all cortical laminae associated with the SIhindlimb representation from a rat that sustained neonatal forelimbremoval. A: CO-stained section through SI cortex. B: PHA-L injectionsite in a section adjacent to that shown in Figure 5A. C: Compositedrawing demonstrating the labeled intracortical fibers in relation tothe SI stump representation. The gray shading represents the injec-tion site and the surrounding intensely stained fibers and cells.Individual fibers are represented as black lines. D: High-powerphotomicrograph showing PHA-L-stained fibers and cells in the SI

hindlimb injection site. E,F: High-power darkfield photomicrographsdemonstrating PHA-L-labeled fibers within dysgranular cortex (E)and the SI stump field (F), respectively. The arrows in E and F showPHA-L-labeled fibers identified in dysgranular cortex and the SIstump area, respectively. The locations from which these photomicro-graphs were taken are indicated by squares labeled D, E, and F in B.For orientation, the anterior (a) and lateral (l) directions are indicatedby the arrows in the top right hand corners of A–C. Scale bar 5 1 mmfor A,B, 10 µm for D–F.

GABA-blocked SI cortex of rats that sustained neonatalforelimb removal may result, at least in part, from changesin its thalamic input.

Technical limitations

The analysis of BDA and PHA-L fiber labeling in thisstudy is dependent on our ability to place correctly thepattern of fiber labeling relative to the CO map of SI andthe degree to which the CO map accurately reflectsthe representations in SI cortex. We were able to positionour BDA injections accurately relative to the CO mapsusing holes left by blood vessel lumens. Additional results

from experiments in which the borders of the forelimband hindlimb representations were marked with TBfurther support our ability to localize BDA labeling tothe hindlimb and forelimb or stump representation.

The results from retrograde labeling of thalamocorticalafferents projecting to the forelimb-stump and hindlimbrepresentations of SI are limited by the fact that ourinjections of TB and DY were not large enough to label allthalamocortical cells projecting to these particular regionsof somatosensory cortex. Thus, each case examines only asample of the overall population of forelimb-stump andhindlimb SI projecting thalamocortical cells. This leaves

Fig. 7. Comparison of the intracortical connections in all corticallaminae projecting from the SI hindlimb representation in neonatallyamputated rats and a normal rat injected with PHA-L. A–C: Compos-ite drawings of three cases in which neonatally amputated rats wereinjected with PHA-L centered in layer IV of SI HL area. D: Case from anormal rat that was also injected in SI HL region. As in Figure 5, the

gray shading represents the injection site and surrounding area ofintense labeling and the more distinct individually labeled fibers arerepresented as black lines. For orientation, the anterior (a) and lateral(l) directions are indicated by the arrows in the top right hand corner ofA. Scale bar 5 1 mm.


open the possibility that we did not label all the cellswithin the hindlimb representation of VPL that projectedto the forelimb-stump representation of SI.

Finally, the percentage of VPL cells physiologicallycharacterized as projecting to the forelimb-stump SI repre-sentation in normal rats and those that sustained neona-tal forelimb removal was lower than anticipated given

that almost all cells within the VB complex project to SI(Saporta and Kruger, 1977). The low percentage of de-tected thalamocortical projecting cells identified in ourrecording experiments is probably associated with the veryshort latencies observed when antidromically firing thesecells and our frequent inability to separate the antidromicresponse from the stimulation artifact.

Fig. 8. Retrograde labeling of SI hindlimb and forelimb-stumpprojecting ventroposterior lateral (VPL) cells with True Blue (TB) andDiamidino Yellow (DY), respectively, in a rat that sustained neonatalforelimb removal and a normal rat. A,B: Photomicrographs of asection from the VPL nucleus of a neonatally amputated rat as seenunder UV-2A and V-2B filters, respectively. C,D: Photomicrographs ofa VPL section from a normal rat viewed under identical filterconditions. For A–D, the dorsal (d) and medial (m) directions areindicated by the arrows in the lower left hand corner of D. UnderUV-2A (A and C) both TB (SI hindlimb projecting, outlined by the black

dots), and DY (SI forelimb-stump projecting) labeled cells are seen astwo relatively separate populations. Under UV-2B (B and D), onlyDY-labeled cells are visible. Note that under UV-2B filter conditions, asmall number of DY-labeled cells are seen among the SI hindlimbprojecting VPL neurons (outlined by the black dots). These cells tendto be located along the medial and ventral margins of the HL SIprojection VPL cell population. This small group of SI forelimb-stumpprojecting VPL cells was counted throughout the extent of VPLnucleus (see Materials and Methods) and the results are depicted inFigure 9. Scale bar 5 500 µm.


Characteristics of intracortical projections

Consistent with the results of Chapin et al. (1987) andFabri and Burton (1991), BDA labeling of intracorticalconnections in our study revealed the following character-istics: 1) the region of SI into which BDA was injected, i.e.,forelimb-stump or hindlimb SI, had the densest fiber andcell body labeling; 2) BDA also sparsely labeled fibers andcells in areas adjacent to the forelimb-stump and hindlimbSI representations characterized as dysgranular cortex(Chapin et al., 1987); and 3) there was little, if any, fiberand cell staining within the forelimb-stump SI when BDAwas injected into the hindlimb SI representation, and viceversa.

The results obtained with PHA-L were consistent withthose obtained using BDA. Taken together, the data fromboth the BDA and PHA-L tracer studies demonstrate twopoints. First, there are normally minimal intracorticalconnections that directly link the forelimb and hindlimb

representations of granular SI, and second, followingneonatal forelimb removal, there is no substantial intracor-tical axonal sprouting from the hindlimb SI area to thestump representation of SI.

Our findings contrast with those of Darian-Smith andGilbert (1994, 1995), who reported that functional changesin the visual cortex of adult cats and monkeys could in partresult from axonal sprouting within cortex and not frommodifications occurring subcortically in the dLGN. How-ever, there are limitations to a comparison between thepresent results and those provided by these investigatorsdue to differences in species age at the time of manipula-tion, the systems being investigated, and the degree offunctional reorganization being examined.

In considering the potential role for intracortical connec-tions in the functional reorganization reported by Lane etal. (1997), it must be noted that our results do noteliminate the possibility that neonatal forelimb removalmay produce modifications in polysynaptic intracorticalconnections not revealed by anterograde and retrogradetracing.

Organization of thalamocortical projections

The present results indicate that neonatal forelimbremoval does not alter the organization of thalamocorticalprojections as revealed by retrograde tracing. The lack ofchange in the animals we studied contrasts with resultsfrom Verney et al. (1982), who reported that thalamocorti-cal fibers projecting to barrel cortex in mice that have beendewhiskered at birth are modified. However, our resultsare consistent with those of McKinley and Kruger (1987),who reported no changes in the thalamocortical projec-tions of cats that sustained T12 spinal lesions at 2 weeks ofage.

It is important to note that our results should not betaken as evidence that peripheral manipulations cannotresult in more subtle changes in thalamocortical afferents.Boxer and Kossut (1985) reported changes in thalamocorti-cal patterning within barrel cortex of rats with all vibris-sae but one removed at postnatal day 1. Furthermore,Jensen and Killackey (1987) and Catalano et al. (1995)have observed changes in individual thalamocortical axonsin barrel cortex in rats following neonatal infraorbitalnerve transection. These changes included disruption ofthe pattern of afferent projections to barrel cortex andwidening of the axonal arborizations of individual neu-rons.

Functional reorganization of VPL

Many investigators have reported reorganization in thesensory thalamus following peripheral deafferentation (Ver-lay and Onnen, 1981; Garraghty and Kaas, 1991; Nicoleliset al., 1991, 1993; Chiaia et al., 1992; Kiss et al., 1994;Rasmusson, 1996). Thus, it was reasonable to hypothesizethat the reorganization seen in SI of rats that sustainedneonatal forelimb removal might be in part due to changesat the level of the thalamus. We identified a small, butsignificant, population of VPL cells (19.0%) that expressedconvergent sensory inputs from both the stump and hind-limb. This is consistent with previous results demonstrat-ing such cells at the level of the CN (Lane et al., 1995) andSI (Lane et al., 1997).

Lane et al. (1995) reported that nearly 70% of CN cells inneonatally amputated rats that were responsive to tactilestimulation of the stump also had a hindlimb component to

Fig. 9. Graph showing the average number of DY-labeled VPL cellsidentified as being part of the SI hindlimb projecting population ofcells within VPL nucleus that project to the SI forelimb-stump area ofcortex in both neonatally amputated rats (n 5 4) and normal rats (n 54). The methods for counting these cells were described earlier and anexample is shown in Figure 8. The bars indicate standard error. Thetwo averages were not significantly different (P . 0.05).

TABLE 1. Functional Properties of VPL Neurons in Adult Rats ThatSustained Neonatal Forelimb Removal1

No.of

ratsTotalunits

No. ofSF

neurons(%)

No. ofSF

cellsthat

projectto FL-stumpSI (%)

No.of DF

neurons(%)

No. ofDF

cellsthat

projectto

StumpSI (%)

Averageresponselatency

(ms)

Neonatallyamputatedrats

10 63 51(81.0)

24/46(52.0)

12(19.0)

4/12(33.3)

BP (DF) 14.4 6 6.5SN (DF) 24.0 6 8.7BP (SF) 11.6 6 4.1

Normal rats 7 54 54(100)

8/21(38.0)

0(0)

—

BP (SF) 7.1 6 3.0

1BP, brachial plexus (stimulation); DF, dual field (FL and HL receptive fields) neuron;SF, single field (FL receptive field only) neuron; SN, sciatic nerve (stimulation).


Fig. 10. Example of a VPL neuron with dual hindlimb and stumpreceptive fields in a rat that sustained neonatal forelimb removal.A: Electrolytic lesion (arrow) within the VPL nucleus (outlined by thewhite dots) used to confirm the location of the single unit recording.B: Receptive fields for the VPL neuron shown in the figure. The stumpreceptive field is indicated by arrow labeled 1 and arrow 2 identifiesthe hindlimb receptive field. C: Response of the VPL neuron toelectrical stimulation of the brachial plexus. D: Response to electricalstimulation of the sciatic nerve. E: Antidromic firing of the neuron bystimulation in the SI stump area. F: Antidromic firing of VPL neuronat a stimulation frequency of 100 Hz. G: Collision data. G1: Firing of

the neuron in response to brachial plexus stimulation. G2: The sameneuron antidromically stimulated with a 7-ms delay. G3: Collisionbetween the ascending response to BP stimulation and the antidromicresponse. Arrows in G2 and G3 identify the artifact from corticalantidromic stimulation. H1: Response of the unit to cutaneous stimu-lation (arrow above the trace) of the stump receptive field (1) shown inB. H2: Response of the unit to cutaneous stimulation (arrow above thetrace) of the hindlimb receptive field (2) shown in B. Vertical scalebar 5 10 mV for C–H. Horizontal scale bar 5 5 ms for C and D, 1 ms forE and F, 2 ms for G, 50 ms for H. Scale bar 5 340 µm.

their receptive fields. In thalamus, this value dropped to19%, and in cortex, only 6% of recording sites in forelimb-stump SI responded to hindlimb stimulation prior toGABA blockade. At both the thalamic and cortical levels,the reduction in recording sites demonstrating both stumpand hindlimb information was significantly reduced com-pared to the preceding level (P , 0.015, ANOVA withScheffe’s post hoc test). Thus, reorganization of the somato-sensory system following neonatal forelimb removal ap-pears to occur at each level of the neuraxis, but theexpression of the change in the CN is progressivelyreduced in the thalamus and cortex. A similar phenom-enon has been reported in monkeys following mediannerve section just prior to and slightly after birth: reorgani-zation is observed in CN, but the functional organization ofthe cortical hand representation remains relatively nor-mal (Florence et al., 1996). The present study, as well asthat of Florence et al. (1996), raises the question: What isthe mechanism(s) underlying the suppression of subcorti-cal reorganization at higher levels of the neuraxis?

The suppression of abnormal hindlimb inputs in neona-tally manipulated rats appears to involve inhibitory mecha-nisms. Lane et al. (1997) have shown that pharmacologicblockade of cortical GABA receptors results in a significantincrease in the expression of hindlimb inputs withinforelimb SI. The decrease in the percentage of stump-hindlimb cells from the CN to VPL strongly suggests thatthe inappropriate hindlimb inputs may also be suppressedat the thalamic level as well. Several potential mecha-nisms could be involved in the decreased expression ofabnormal hindlimb inputs to the VPL. Inhibitory circuitsinvolving GABA could be involved in the suppression of theabnormal hindlimb-stump information within the VPLstump representation in neonatally amputated rats. Al-though there appear to be no GABAergic interneuronswithin the VB nuclei of rats (Barbaresi et al., 1986; Harrisand Hendrickson, 1987; Bentivoglio et al., 1991), thalamo-cortical cells send collateral projections to the nucleusreticularis thalami (nRT; Jones, 1975; Harris, 1987), whichis composed of neurons that project back to the VPL andutilize GABA as a neurotransmitter (Houser et al., 1980;Montero and Singer, 1985; de Biasi et al., 1986; Spreaficoet al., 1991). Therefore, it is possible that the aberranthindlimb information that reaches VPL is being sup-pressed via feedback inhibitory mechanisms involvingnRT. Such a hypothesis is supported by reports of Lee et al.(1994a,b), which showed that nRT is involved in the activeregulation of functional properties (including suppressionof receptive fields) of ventroposterior medial nucleus (VPM)neurons in rats.

Another pathway that might participate in VPL suppres-sion of hindlimb information involves descending corticofu-gal fibers, which send projections to both VPL and nRT(Jones, 1985). How corticofugal fibers participate in theoverall activity of VPM or VPL nuclei is still unclear. Someinvestigators have reported that corticothalamic axonsinhibit thalamic cells (Burchfiel and Duffy, 1974; Marroccoet al., 1982; Shin and Chapin, 1990), whereas others havedescribed these fibers as having a net excitatory effect onsomatosensory thalamus (Schmielau and Singer, 1977;Yuan et al., 1985, 1986; Villa et al., 1991; Diamond et al.,1992; Ghosh et al., 1994). Still others suggested thatcorticofugal projections have a combination of both inhibi-tory and excitatory influences in the somatosensory thala-mus (Tsumoto et al., 1975).

Changes in response latencies in VPL

The functional reorganization of VPL in neonatallymanipulated rats was accompanied by an increase in theresponse latency of VPL cells to brachial plexus stimula-tion. Response latency data (unpublished observations)from CN neurons of neonatally manipulated rats studiedby Lane et al. (1995) show a similar trend: the averageresponse latency of CN neurons with stump-hindlimbreceptive fields to brachial plexus stimulation was signifi-cantly longer when compared with normal CN cells (6.30 66.90 ms vs. 2.93 6 1.09 ms, P , 0.001). Although notsignificant (P . 0.05), the average response latencies ofsingle-field CN neurons in neonatally manipulated rats(3.57 6 1.63 ms) was longer when compared with normalCN neurons. It was also shorter than that for CN cells withdual receptive fields, although not significantly (P . 0.05).

Longer response latencies have also been reported forVPM (Chiaia et al., 1992; Nicolelis et al., 1997) and corticalneurons (Waite, 1984) following neonatal infraorbital nervetransection. The most likely explanation for the increasedresponse latencies reported here is that neonatal forelimbamputation produces morphologic and functional changesin primary afferents that survive neonatal manipulation.Previous work in young kittens (Risling et al., 1980, 1983;Aldskogius and Risling, 1981) and neonatal rats (Bondakand Sansone, 1984) suggests that the primary afferentsthat survive peripheral nerve injury have smaller axondiameters and thinner myelin sheaths than normal pri-mary afferents. Of particular interest is a report by Heathet al. (1986) that examined the effects of neonatal forelimbamputation in rats. They reported that 4 weeks afteramputation, the surviving C6 sensory afferents showeddecreased axon size and thickness in myelin sheaths. Ifsuch changes persist into adulthood, it suggests that thesensory afferents that survive neonatal forelimb removalshould have slower conduction velocities and result inlonger response latencies as observed in this study.

In summary, the results of this study demonstrate thatthe functional reorganization seen in the somatosensorycortex of rats that sustained neonatal forelimb removalmay be due in part to functional changes occurring atsubcortical levels, in particular, thalamic VPL.

ACKNOWLEDGMENTS

Special thanks to Rebecca Wynn for her excellent techni-cal assistance and Dr. Carol Bennett-Clarke for her helpfulcomments and suggestions.

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thalamocortical and intracortical projections to the forelimb-stump si representation of rats that...

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