Corticocuneate projections are altered after spinal cord dorsal column lesions in New World monkeys

Abstract

Recovery of responses to cutaneous stimuli in the area 3b hand cortex of monkeys after dorsal column lesions (DCLs) in the cervical spinal cord relies on neural rewiring in the cuneate nucleus (Cu) over time. To examine whether the corticocuneate projections are modified during recoveries after the DCL, we injected cholera toxin subunit B (CTB) into the hand representation in Cu to label the cortical neurons after various recovery times, and related results to the recovery of neural responses in the affected area 3b hand cortex. In normal New World monkeys, labeled neurons were predominately distributed in the hand regions of contralateral areas 3b, 3a, 1 and 2, parietal ventral (PV), secondary somatosensory cortex (S2), and primary motor cortex (M1), with similar distributions in the ipsilateral cortex in significantly smaller numbers. In monkeys with short-term recoveries, the area 3b hand neurons were unresponsive or responded weakly to touch on the hand, while the cortical labeling pattern was largely unchanged. After longer recoveries, the area 3b hand neurons remained unresponsive, or responded to touch on the hand or somatotopically abnormal parts, depending on the lesion extent. The distributions of cortical labeled neurons were much more widespread than the normal pattern in both hemispheres, especially when lesions were incomplete. The proportion of labeled neurons in the contralateral area 3b hand cortex was not correlated with the functional reactivation in the area 3b hand cortex. Overall, our findings indicated that corticocuneate inputs increase during the functional recovery, but this functional role is uncertain.

Graphical Abstract

The cuneate nucleus normally receives cortical inputs from the hand regions of sensorimotor cortex in the contralateral and ipsilateral hemispheres. This cortical descending influence remains similar in New World monkeys with the short recoveries from the dorsal column lesion, but is greatly increased after the long recoveries.

INTRODUCTION

Dorsal column lesions (DCLs) in the cervical spinal cord deprive the cuneate nucleus (Cu) in the brainstem of most or all of the primary activating afferents from the hand, resulting in the neural inactivation of subsequent levels in the hand representations of the contralateral ventroposterior nucleus in the thalamus and primary somatosensory cortex (S1, or area 3b in monkeys). After weeks to months of recovery, the affected hand neurons in area 3b largely become responsive to touch on hand, and the somatotopy that had been disturbed by the lesion returns to nearly normal in some cases (Cheng et al., 2015; Jain et al., 2000; Jain et al., 2008; Qi et al., 2011; Qi et al., 2014). Such plastic changes in the affected hand region in area 3b are primarily dependent on the rewiring of remaining connections to and within the Cu (Kambi et al., 2014; Liao et al., 2016; Xu and Wall, 1999a). The rewiring involves an interplay between surviving primary afferents and second-order spinal cord afferents that become more effective in conveying the cutaneous inputs from the hand (Liao et al., 2015; Liao et al., 2018), the intranuclear circuit involving the excitatory and inhibitory neurons, and the descending cortical projections (Aguilar et al., 2003; Andersen et al., 1964; Catsman-Berrevoets and Kuypers, 1976; Cheema et al., 1985; Cheema et al., 1983; Lue et al., 2001; Lue et al., 1997).

Several cytoarchitectonic areas of sensorimotor cortex send axonal projections to the contralateral Cu in a somatotopic pattern in macaque monkeys (Bentivoglio and Rustioni, 1986; Cheema et al., 1985; Coulter and Jones, 1977) and in squirrel monkeys (Wise and Jones, 1977). Anterograde tracing and degeneration methods showed that the area 3b hand region projects focally to the dorsal part of Cu, especially in the middle segment that is known as the “core” or “pars rotunda” (Cheema et al., 1985; Kuypers, 1958). This part of Cu is heavily innervated by low-threshold cutaneous inputs from the palm and digits of the hand, and it projects to the hand representation in the contralateral ventroposterior thalamic nucleus (Florence et al., 1991; Keller and Hand, 1970; Kuypers and Tuerk, 1964; Shriver et al., 1968; Xu and Wall, 1999b). The cortical inputs from areas 1 and 2 to the Cu are present throughout most of the nucleus but are particularly dense in the rostral sector (Cheema et al., 1985). In addition, the area 3a and primary motor cortex (M1) project mainly to the ventral and lateral fringe of middle and caudal Cu and the external cuneate nucleus (ECu) (Cheema et al., 1985; Kuypers, 1958), which are largely innervated by proprioceptive inputs from the proximal forelimb, shoulder and neck (Hummelsheim and Wiesendanger, 1985; Hummelsheim et al., 1985; Witham and Baker, 2011). Retrograde tracer injections in the Cu added to these results by showing that the corticocuneate projection neurons are located in the layer V of areas 3b, 1 and 2, and M1, and additional cortical areas, including the secondary somatosensory cortex (S2), parietal ventral (PV), premotor cortex (or area 6), posterior parietal cortex (or areas 5 and 7), supplementary motor cortex (SMA) and cingulate cortex (Cg) (Bentivoglio and Rustioni, 1986; Catsman-Berrevoets and Kuypers, 1976; Cheema et al., 1985; Wise and Jones, 1977).

The cortical projections, especially from S1 (area 3b), to the contralateral Cu serve to augment the neural response and sharpen the receptive fields of ongoing sensory processing for neurons in the Cu for accurate cutaneous discriminations (e.g., Aguilar et al., 2003). However, whether and how this corticocuneate modulation is involved in the functional recovery that occurs after the sensory loss following spinal cord injury remains unknown. As the cortical reactivation process can result in some disorganization of the somatotopy of the cortical hand representation, and neurons with larger than normal receptive fields (Reed et al., 2016), the cortical projection to the Cu could be altered in ways that compensate for this altered cortical somatotopy. Thus, cortical influences may be preserved, or even expanded, to strengthen the signaling transmission from the spared spinal inputs to the Cu, which would be reflected at the higher-order processing levels in the cortex. Or, the cortical modulation may be weakened by losing the connections over time because the driving source of the lemnisco-thalamocortico-cuneate loop has been interrupted by the spinal cord injury. Here we tested for these possibilities by studying the anatomical organizations of corticocuneate connections in normal monkeys, and monkeys with the DCLs at short-term (2 weeks) or long-term (over 7 months) recovery periods, and related these connections to the extent of cortical reactivation in the hand region of area 3b. We used squirrel and owl monkeys as the experimental animals to allow comparisons of the current results to our previous spinal cord injury studies in these monkeys. Thus, we made unilateral DCLs at the spinal cord C4-C5 levels. After 2 weeks or more than 7 months of recovery, a retrograde tracer, cholera toxin subunit B (CTB), was injected into the electrophysiologically-defined Cu ipsilateral to the DCL in lesioned monkeys to label the projection neurons at the cortical level. Injections into the Cu of normal monkeys were used as a control. The extents of cortical reactivation of the hand region of area 3b contralateral to the DCL were evaluated by microelectrode recordings.

The results indicate that cortical connections from the contralateral and ipsilateral hemispheres to the deprived Cu are largely preserved after the short-term DCLs, although we observed a slight decrease of labeled neurons in the area 3b hand cortex. However, a more widespread distribution of labeled neurons is present across cortical areas after the Cu injection in monkeys with the longer recoveries. Possibly, the increased cortical projections from several sensorimotor cortical areas of the two hemispheres contributed to the recovery process.

MATERIALS AND METHODS

Nine New World squirrel monkeys (6 Saimiri boliviensis; 3 Saimiri sciureus) and four owl monkeys (Aotus nancymaae) were used in this study. The two species have highly-similar organizations of somatosensory system (Kaas, 2004; Liao et al., 2015; Merzenich et al., 1978; Sur et al., 1982). All surgical procedures and animal care were conducted in accordance with the Guides for the Care and Use of Laboratory Animals by the National Institutes of Health and Vanderbilt University. Monkeys were divided into a normal group (n = 3; OM-L, OM-B, SM-Y; see Table 1), a short-term DCL group (n = 4; SM-Rol, SM-Soo, SM-Ru, SM-LM), and a long-term DCL group (n = 6; SM-D, SM-P, SM-W, SM-Rog, OM-St, and OM-A) depending on the recovery times. Within the two DCL groups, animals were further divided into a subgroup with nearly complete lesions (>95% lesion extent) and a subgroup with less complete lesions (<95% lesion extent). Monkeys OM-St and OM-A had the DCL at the C5 level, and all other monkeys received the DCL at the C4 level. The somatotopic maps from 7 monkeys were previously reported (Liao et al., 2015; Liao et al., 2018), but the results of the labeling of corticocuneate neurons from all cases were not previously published.

Table 1.

Case summary

				Normal group
Case	Gender	Species	Les. Level	Les. Ext. (%)	Recovery Time	Cutting Plane
OM-L	female	A. nancymaae	N/A	N/A	N/A	flattened
OM-B	female	A. nancymaae	N/A	N/A	N/A	flattened
SM-Y	male	S. boliviensis	N/A	N/A	N/A	flattened
				Short-term DCL
SM-Rol	male	S. boliviensis	C4	91%	14d	flattened
SM-Soo	female	S. boliviensis	C4	91%	14d	flattened
SM-Rue	female	S. boliviensis	C4	100%	14d	coronal
SM-LM	female	S. boliviensis	C4	97%	14d	flattened
				Long-term DCL
SM-D	female	S. sciureus	C4	87%	209d	flattened
SM-P	female	S. sciureus	C4	76%	245d	coronal
SM-W	female	S. sciureus	C4	100%	251d	flattened
SM-Rog	male	S. boliviensis	C4	100%	231d	flattened
OM-St	male	A. nancymaae	C5	96%	297d	flattened
OM-A	female	A. nancymaae	C5	97%	302d	flattened

General surgical procedure

Monkeys were initially tranquilized with an intramuscular injection of ketamine hydrochloride (10–25 mg/kg) and anesthesia was maintained by isoflurane (1–2% mixed in O₂) during the surgical procedures. All procedures were performed under aseptic conditions and vital signs including heart rate, blood pressure, oxygen saturation (spO₂), body temperature, and respiration rate were monitored every ten minutes throughout the procedure. The anesthesia was switched to intravenous injections of ketamine hydrochloride (10–25 mg/kg) during the electrophysiological mapping. In survival surgeries involving the Cu and cervical spinal cord, the monkey’s head was positioned downward with neck flexion to facilitate the exposure of the magnum foramen and cervical vertebra. Each monkey’s recovery from anesthesia was closely monitored. In the terminal recording sessions, the monkey’s head was fixed in a standard stereotaxic position for the access to the somatosensory area 3b in the parietal cortex.

Unilateral dorsal column lesion

After an incision was made in the skin at the midline over the neck, the muscle layers above the C3–C5 vertebrae were retracted, the dorsal arch of the C4–C5 or C5–C6 vertebra was removed, and the dura and pia that covered the exposed spinal cord were displaced. We used fine-tipped forceps to crush the unilateral dorsal columns at C4 or C5 for 2 minutes. This crush was followed by a cut with surgical micro-scissors at the same location. The exposed spinal cord was then protected by a piece of Gelfoam before the opening was closed. While specific task performance was not tested in any of these monkeys, observations of cage behavior revealed that deficits in hand use appeared immediately after the lesion, and could last for days. No locomotion was hampered by the DCL, and hand use in most monkeys returned to nearly normal within weeks to few months (see Qi et al., 2013).

Microelectrode mapping and tracer injection in the Cu

In both normal monkeys and monkeys with DCLs, responsiveness to tactile stimulation of the hand in the Cu was recorded to determine the tracer injection site. Two weeks before the terminal procedure, one retrograde tracer, cholera toxin subunit B (CTB; 1% in dH₂O; Catalog no. C9903, Sigma, St. Louis, MO), was injected into the electrophysiologically-defined hand representation in Cu to label the projection neurons in the cortex. In monkeys with the short-term DCL, the hand region of Cu was mapped and the CTB injection was placed immediately before the DCL in the same surgery. The short-term recovery period was two weeks without a separate surgery. After the brainstem was exposed, a single tungsten microelectrode (1MΩ) was perpendicularly inserted into the Cu (see details in Liao et al., 2015, 2018). Standard mapping procedures, including lightly touching and brushing the skin, tapping the muscles, and moving the joints, were used to identify the neural receptive fields and response strengths. Strong responses to touch on the digits were recorded in all normal monkeys and in pre-lesion recordings in short-term lesioned monkeys, and responses to the digits, wrist and arm were also recorded in the post-lesion recordings from monkeys with long recoveries. The normal somatosensory organization of Cu in New World squirrel monkeys (Xu and Wall, 1999b) was also considered to determine the injection site. Once the location of the representation of hand in Cu was identified, 0.05 – 0.1μl of CTB was injected at two depths, 800 and 600 μm, below the pia surface. After the injection micropipette was withdrawn, the exposed area was covered by Gelfoam and/ or Gelfilm, and the opening was closed.

Subcutaneous digit injection

To estimate the lesion extent, CTB conjugated with wheat germ agglutinin-horseradish peroxidase (B-HRP; 5 μl, 0.2% in distilled water; Catalog no. C34780, Invitrogen, Carlsbad, CA) was injected into the matching parts of digits 1, 3, and 5 of both hands in most monkeys. Injections were placed 5 days before the terminal procedure to allow tracer transportation from the skin to the spinal cord and brainstem Cu (Liao et al., 2015; Qi et al., 2011).

Microelectrode mapping in the area 3b

Two weeks after the Cu injection, neural responses in the hand region of area 3b contralateral to the DC lesion were examined with multiunit microelectrode recordings. A low-impedance tungsten microelectrode was perpendicularly lowered from the cortical surface to a depth of 650 μm, where the layer IV is located. We systematically placed microelectrode penetrations every 300–400 μm across the surface of the expected location of hand region in area 3b. To define the mediolateral borders of the hand region of area 3b, we recorded from neurons in at least one additional row of electrode penetrations across the cortex where neurons responded to touch on the face or the arm lateral or medial to the normal hand region. Standard mapping procedures were used to identify the neuronal receptive fields (e.g., Merzenich et al., 1978) . At the conclusion of the mapping, small electrolytic lesions were made with a continuous current at 10 μA while the recording electrode was withdrawn from a depth of 2 mm to the brain surface. The marking lesions were placed at the rostral and caudal borders of the hand region in area 3b for identification after tissue processing.

Perfusion and histology

The monkeys were euthanized with a high dose of sodium pentobarbital (120 mg/kg). After death, monkeys were perfused through the ascending aorta with 0.1M phosphate buffered saline (PBS; pH 7.4), followed by 2–4% paraformaldehyde in 0.1M phosphate buffer (PB) and 10% sucrose-containing fixative. The brain and spinal cord (C2–C8) were removed and saved in 30% sucrose-PB for cryoprotection. We placed pins at the boundaries between individual cervical spinal cord segments based on the rostrocaudal arrangement of dorsal rootlets, thus, we could identify the cervical segments after the tissue was cut. The brainstems from all monkeys were cut in the transverse plane at a thickness of 50 μm, and the spinal cords were cut in the horizontal plane at 40 μm or in the transverse plane at 50 μm. In two monkeys (SM-Ru, SM-P), the brains were cut coronally at a thickness of 50 μm to reveal the laminar distribution of projection neurons. In three monkeys (SM-Y, SM-Soo, SM-LM), the cortex contralateral to the lesion was separated from the subcortical structures and flattened (see details in Gharbawie et al., 2011; Krubitzer & Kaas, 1993; Sincich et al., 2003). In the other eight monkeys, cortex from both hemispheres were flattened. All flattened cortex were cut parallel to the brain surface at a thickness of 40 μm. The sections of each tissue block were divided into series for specified histological procedures to reveal the CTB labeling (Angelucci et al., 1996; Liao et al., 2013), B-HRP labeling (3,3,5,5,-tetramethylbenzidine reaction, TMB; Gibson et al., 1984), and architectural structures with cytochrome oxidase (CO; Wong-Riley, 1979) and vesicular glutamate transporter 2 (VGLUT2). See details in previous publications (Liao et al., 2015; Liao et al., 2016; Liao et al., 2018).

Antibody characterization

Anti-vesicular glutamate transporter 2 (VGLUT2) primary antibody (AB_ 2187552) is a mouse anti- VGLUT2 recombinant protein (monoclonal; Catalog no. MAB5504, Millipore, Burlington MA). The immunogen is a KLH-conjugated linear peptide. In western blots of primate neocortex, the antibody recognizes a 56-kDA bond, the molecular weight of VGLUT2 (Baldwin et al., 2013). This primary antibody was used in a concentration of 1:5000.

Anti-NeuN primary antibody (AB_2298772) is a mouse IgG1 antibody with clone A60 (monoclonal; Catalog no. MAB377, Millipore). It was prepared against purified cell nuclei from mouse brain and specifically recognizes the DNA-binding, neuron-specific protein NeuN in most central and peripheral nervous system (CNS and PNS) neuronal cells in vertebrates including primates (Balaram and Kaas, 2014; Rovo et al., 2012). This antibody labels 2–3 bands in the 46–48 kDa range and possibly another band at 66 kDa in western blot preparation (adapted from product information). This primary antibody was used in a concentration of 1:5000.

Data analysis

Evaluation of lesion level and extent

In most monkeys with the DCL, the lesion level was identified in sections of the spinal cord based on the pin placements between the cervical segments, and was confirmed by the B-HRP labeled patches at C5, C6, and C7 from tracer injections in digits 1, 3, and 5 (Florence et al., 1991). In monkeys that had the spinal cord cut in the horizontal plane, a transverse view of the DCL was reconstructed from a series of horizontal sections. The difference in areas of B-HRP in the Cu on the intact and lesioned sides was used to estimate the lesion extent. After the brainstem sections were processed for B-HRP labeling, the areas of B-HRP labeled patches in Cu on both sides across the rostrocaudal brainstem sections were measured using the ImageJ 64 software (National Institutes of Health, Bethesda, MD). Values from individual sections were summed to obtain the total labeled area of total B-HRP labeling on the lesioned side and intact side. In cases with the DCL restricted to the lesioned side, the total area of B-HRP labeled terminals in the Cu on the intact side was used as the control. The percentage of reduced B-HRP labeled area on the lesioned side to the total B-HRP labeled area on the intact side was used as a quantitative estimate of the effective DCL extent in each monkey (Qi et al., 2011; Liao et al., 2016). Or, the estimated DCL extent = [(summed B-HRP labeling value on the intact side – summed B-HRP labeling value on the lesioned side)/ summed B-HRP labeling value on the intact side] × 100%. However, in cases where the lesions involved at least part of the dorsal columns of both sides, some of the dorsal column projections from the hand to the Cu on the intact side were also interrupted by the lesion, hence the total B-HRP labeled area in the Cu cannot be used as the control. Lesion extents were estimated by calculating the percentage of the size of the lesion in the dorsal columns to the size of dorsal columns on the same side.

Identification of corticocuneate connections

The location of the CTB injection site in the brainstem and distributions of CTB-labeled neurons in the cortex were systematically plotted using the Neurolucida system (MBF Bioscience, Williston, VT). Special care was taken to mark blood vessels, landmarks, and electrolytic lesion sites for the alignment of plotted label to the adjacent CTB-labeled cortical sections, and the adjacent VGLUT2 sections that revealed the areal borders and the hand/ face border. Based on the receptive fields and response modalities of each recording site, the sensory maps of the hand representations of areas 3b and 1 were reconstructed (Liao et al., 2015; Liao et al., 2013; Liao et al., 2018). The borders of the area 3b hand region were estimated based on the mapping results and the architectural information revealed by the VGLUT2 sections. The estimated locations and widths of areas 3a, 1, and 2 were based on previous publications, as was the width of M1 (Card and Gharbawie, 2020; Dancause et al., 2006; Gould et al., 1986; Preuss et al., 1996; Stepniewska et al., 2014; Stepniewska et al., 1993, 2006; Wu and Kaas, 1999). The estimated locations of other cortical areas are relative to area 3b and cortical fissures. The mediolateral extents of hand regions in areas 3a, 1 and 2 were approximately located in parallel to the hand region of area 3b (Merzenich et al., 1978; Sur et al., 1982). However, the microstimulation-identified representations of hand and arm in M1 are intermingled (Preuss and Kaas, 1996; Wu and Kaas, 1999), forming a forelimb region in parallel to the hand-arm region of primary somatosensory cortex.

Percentage of cortical inputs to the Cu

We first examined whether the percentages of cortical inputs from the contralateral and ipsilateral hemispheres to the deprived Cu are altered at the short- and long-term recovery periods after various extents of the DCLs. The data were obtained from 10 cases including two normal monkeys, one monkey with the short-term and incomplete (<95%) DCL, one monkey with the short-term and nearly complete (>95%) DCL, two monkeys with long-term and incomplete DCLs at C4, two monkeys with long-term and nearly complete DCLs at C4, and two monkeys with the long-term and nearly complete DCL at C5 (Table 2). The percentages of projections from each hemisphere were calculated by the number of labeled neurons in one hemisphere divided by the number of total labeled neurons in the two hemispheres. The percentage of hemispheric projections = (the number of labeled neurons in contralateral or ipsilateral hemisphere/ the number of total labeled neurons in the two hemispheres) × 100%. Next, whether the numbers of labeled neurons in different sensorimotor areas in the contralateral cortex were altered were examined. We calculated these percentages of labeled neurons in the expected locations of hand regions of area 3b, 3a, 1 and 2 (1/2), the forelimb region of M1, and PMd, PMv, the cortex of lateral sulcus (PV/S2, PR, Ri, and VS), and others in the contralateral cortex for each monkey. That is, the percentage of projections by area = (the number of labeled neurons in selected cortical regions and areas of the contralateral cortex/ the number of total labeled neurons in the contralateral cortex) × 100%.

Table 2.

Percentage of labeled neurons in two hemispheres

Case	contralateral	ipsilateral
Normal
OM-L (3291)	91.8	8.2
OM-B (2961)	77.6	22.4
SM-Y	*N/A
short-term, <95% DCL at C4
SM-Rol (2691)	96.4	3.6
SM-Soo	N/A
short-term, >95% DCL at C4
SM-Rue (1834)	88.9	11.1
SM-LM	N/A
long-term, <95% DCL at C4
SM-D (5008)	94.9	5.1
SM-P (1466)	93.4	6.6
long-term, >95% DCL at C4
SM-W (4064)	79.7	20.3
SM-Rog (3794)	95.3	4.7
long-term, >95% DCL at C5
OM-St (6230)	81.3	18.7
OM-A (7203)	85.1	14.9

^*Not available due to the lack of data from the ipsilateral cortex.

We particularly focused on changes in the percentage of corticocuneate projection neurons in the hand region of area 3b in the contralateral cortex after the Cu injection. This is because previous studies revealed that the projection from S1 neurons facilitated the signaling transmission in Cu neurons with overlapping receptive fields (Aguilar et al., 2003). Considering that tracer injection size and transportation efficiency may differ across cases, we evaluated the ratio of the labeled projection neurons in monkeys with the DCL versus the normal proportion. First, the average percentages of labeled neurons in the area 3b hand cortex of the contralateral cortex from three normal monkeys was used as the normal projection. Next, the proportion of the corticocuneate projection from the area 3b hand region in the lesioned monkeys was compared to the average proportion in normal monkeys. The ratio of the DCL projection to normal = the percentage of labeled neurons in area 3b hand cortex from the contralateral hemisphere/ the normal projection (the mean value from three normal monkeys). Similarly, the number of recording sites with neurons that responded to touch on the hand was represented as a percent of the total of the recording sites for each monkey, because the total number of microelectrode penetrations collected differed across cases. Thus, the percentage of area 3b hand reactivation = (the number of microelectrode penetrations in area 3b hand cortex with neurons that responded to touch on the hand/ the number of total microelectrode penetrations in area 3b hand cortex) × 100%. We estimated the relationship of corticocuneate projection and the cortical reactivation in the hand cortex using the Pearson correlation (two sided, with 95% confidence interval, using JMP software, Cary, NC). The same strategy was used to evaluate the relationship between the DCL extent and cortical reactivation. While the number of monkeys in each group is small, these correlations were performed without regard to group assignment for all 10 DCL monkeys studied. Figures indicate group category of each monkey to highlight observable trends.

RESULTS

Our primary goal was to determine whether the cortical projections to the deprived Cu are altered after spinal cord injury, thereby affecting the cortical reactivation in the hand region of area 3b, especially after longer recovery times. First, results from normal monkeys are described. Next, we focused on determining the distributions and numbers of labeled cortical neurons in sensorimotor cortex that are contralateral and ipsilateral to the deprived and injected Cu. Third, we analyzed whether the proportion of labeled neurons in the hand region of area 3b is related to the functional neural reactivation in the affected hand cortex. Fourth, the results from the DCL monkeys are described and related to the severity of DCL (<95% vs. >95%), the recovery times (2 weeks vs. >7 months), and the lesion level (C4 vs. C5). In all cases, CTB was injected into the representations of hand in Cu after the electrophysiological recording. The cores and dense uptake zones of CTB injections were located within the territory of Cu and extended approximately 1– 1.5 mm rostrocaudally, occasionally with slight spreading beyond the border.

Projections to Cu in normal monkeys

The normal organization of corticocuneate connections was examined in three monkeys without DCLs. In monkey OM-L (Fig. 1a), CTB was injected into the hand region of Cu on the right side with the core slightly spreading beyond the ventral border of the Cu. Large numbers of CTB-labeled neurons were found in the contralateral cortex including the areas 3b, 3a, 1 and 2, and M1 (Fig. 1b). While most of the labeled neurons were confined to the hand regions of somatosensory areas, clusters of labeled neurons were in adjacent regions of cortex representing the arm and face. The finding of labeled neurons in the arm region was somewhat expected since the representation of arm is adjacent to the representation of hand in Cu and could be somewhat involved by the injection. While the injection core did not spread to the principle trigeminal nucleus that represents the face (see Fig. 1a), the injection halo may have slightly spread to the adjacent spinotrigeminal nucleus and labeled the connection for face. Clusters of CTB-labeled neurons were also located in the regions of the area parietal ventral (PV), second somatosensory area (S2), parietal rostral area (PR), retroinsular cortex (Ri), and ventral somatosensory area (VS) along the lateral sulcus. Labeled neurons were also in the forelimb region of M1, and dorsal (PMd) and ventral (PMv) premotor cortex, and the supplementary motor area (SMA). Labeled neurons in the PV, S2, and VS regions could have been located in the portions representing the arm and hand, but without receptive field mapping, this is uncertain. Many neurons in Cg, frontal, and insular areas of cortex were also labeled. A considerable number of CTB-labeled neurons was found in the right cortical hemisphere ipsilateral to the Cu injection (8.2 % of the total labeled neurons in the two hemispheres; Fig. 1c, Table 2). While slightly more CTB-labeled neurons were distributed in the ipsilateral SMA, Cg and the cortex of the lateral sulcus, small numbers of labeled neurons were identified in the hand-arm regions of areas 3a and M1. In strong contrast to the contralateral hemisphere, only 1 labeled neuron was found in the area 3b, and no labeled neurons were seen in areas 1 and 2 in this case.

Figure 1.

Distribution of labeled neurons in the flattened cortex after CTB was injected into the hand region of cuneate nucleus (Cu) of the normal monkey OM-L. (a) Photomicrograph showing that the injection core and halo (dark and light blue shadings) primarily involved the Cu, slightly expanding beyond its ventral border. (b) The distribution of CTB-labeled neurons (dark blue dots) in the cortex contralateral to the Cu injection. The CTB-labeled neurons are extensively distributed in the contralateral areas 3b, 3a, 1 and 2, primary motor cortex (M1), the parietal ventral (PV) and second somatosensory cortex (S2). Although CTB-labeled neurons are primarily confined to the hand region in these cortical areas, many labeled neurons are in face, arm and trunk regions. Scatterings of CTBlabeled neurons are shown in the expected locations of premotor cortex dorsal and ventral (PMd and PMv), ventral somatosensory cortex (VS), parietal rostral (PR), and retroinsular (Ri) cortex. Additional labeled cortical areas include the supplementary motor area (SMA), cingulate area (Cg). (c) A small number of CTB-labeled neurons are present in the cortex ipsilateral to the Cu injection, predominately in the hand-arm (forelimb) region of area 3a and M1, the cortex of the lateral sulcus, PMd, and Cg. Note that the area 3b hand region has no CTB-labeled neurons. CgS, cingulate sulcus; LS, lateral sulcus. Scale bar is 1 mm in (a), and 5 mm in (b-c).

Similar labeling patterns were observed in the other 2 control monkeys OM-B and SM-Y (Figs. 2 and 3), although some differences in distribution density across cortical regions were present (see percentage of labeled neurons in cortical areas in Table 3). Overall, the Cu injection normally labeled abundant neurons in the contralateral cortex. The labeled neurons are typically located in the areas 3b, 3a, 1 and 2, PV/S2, as well as other somatosensory areas along the lateral sulcus, and several motor areas including the M1, PMd, PMv, and SMA. The relatively smaller number of labeled neurons in the hand region in area 3b in OM-B may have resulted from the small CTB injection site that was primarily located in more medial region of Cu, where responses to touch on digits 4 and 5, palm, wrist, and forearm arm were recorded (also see normal somatotopic map of Cu in Xu and Wall, 1999b; Fig. 2). We also noted many CTB-labeled neurons in the Cg along the medial wall. A significantly smaller number of CTB-labeled neurons were distributed in the cortex ipsilateral to the Cu injection. These labeled neurons were more common in the sensorimotor areas in the medial wall, Cg, and the cortex of the lateral sulcus, with lower numbers in the hand-arm regions of areas 3a and M1. Only occasionally were labeled neurons seen in areas 3b, 1 and 2. The dense distributions of labeled neurons in area 3a of SM-Y indicates that the injection site included some of the ECu (Fig. 3). No obvious differences in the organization of corticocuneate projection from these two normal owl monkeys and one normal squirrel monkey were noted. In these and in lesioned monkeys in which cortex was flattened and cut parallel to the surface, labeled neurons were restricted to the deeper sections, likely layer V. In brains cut in the coronal plane (SM-Rue, SM-P), labeled neurons were confirmed to be in layer V.

Figure 2.

Distribution of labeled neurons in the flattened cortex after CTB was injected into the hand region of Cu of the normal monkey OM-B. (a) Photomicrograph showing that the CTB injection core and halo are located in the medial part of Cu. (b) The distribution of CTB-labeled neurons in the cortex contralateral to the Cu injection. (c) A small number of CTB-labeled neurons are present in the cortex ipsilateral to the Cu injection. Scale bar is 1 mm in (a), and 5 mm in (b-c). Other conventions as in Figure 1.

Figure 3.

Distribution of labeled neurons in the flattened cortex after CTB was injected into the hand region of Cu of the normal monkey SM-Y. (a) Photomicrograph showing the Cu injection. (b) The distribution of CTB-labeled neurons in the cortex contralateral to the Cu injection site. Scale bar is 1 mm in (a), and 5 mm in (b). Other conventions as in Figure 1.

Table 3.

Percentage of labeled neurons in individual cortical area of the contralateral hemisphere

Case	3b-#H	3a-H	1/2-H	M1-##F	PMd	PMv	LS	Other
Normal
*OM-L (3022)	10.6	5.2	5.4	9.9	0.1	0.8	32.5	35.5
OM-B (2298)	3.0	1.3	4.4	6.9	2.0	1.5	20.3	60.6
SM-Y (3238)	8.6	12.8	11.5	13.2	1.1	3.2	17.5	32.1
Average	7.4	6.4	7.1	10.0	1.1	1.8	23.4	42.7
Short-term, < 95% DCL at C4
SM-Rol (2593)	15.9	19.1	14.0	12.0	0.7	3.5	13.6	21.2
SM-Soo (3348)	5.3	9.8	8.2	11.0	1.2	5.3	23.4	35.8
Average	10.6	14.5	11.1	11.5	1.0	4.4	18.5	28.5
short-term, > 95% DCL at C4
SM-Rue (1631)	8.9	11.0	6.6	7.7	0.4	0.7	14.2	50.5
SM-LM (1600)	6.0	10.4	13.0	15.2	4.2	2.8	20.9	27.5
Average	7.5	10.7	9.8	11.5	2.3	1.8	17.6	39.0
long-term, < 95% DCL at C4
SM-D (4753)	8.1	10.2	7.4	15.7	2.5	4.2	18.7	33.2
SM-P (1369)	9.6	8.1	8.1	11.5	0.8	0.1	20.4	41.4
Average	8.9	9.2	7.8	13.6	1.7	2.2	19.6	37.3
long-term, > 95% DCL at C4
SM-W (3237)	10.3	3.6	17.3	3.0	0.8	3.9	15.2	45.9
SM-Rog (3616)	9.0	4.2	11.7	9.5	0.5	4.1	21.3	39.7
Average	9.7	3.9	14.5	6.3	0.7	4.0	18.3	42.8
long-term, > 95% DCL at C5
OM-St (5062)	5.1	4.6	3.8	6.7	1.3	1.9	36.2	40.4
OM-A (6130)	5.5	6.6	7.3	9.9	1.8	1.2	26.6	41.1
Average	5.3	5.6	5.6	8.3	1.6	1.6	31.4	40.8

^*case (number of total labeled neurons)

^#H, hand region

^##F, forelimb region

Short-term recovery group

Incomplete DCLs at C4

To reveal the organization of corticocuneate connections at the subacute phase of spinal cord injury (defined by Rowland et al., 2008), we analyzed the distribution of labeled cortical neurons in monkeys at two weeks after the DCL and Cu injection. Cortical projections at this early time during functional recovery were evaluated by relating the connectional patterns to the reactivated somatotopic maps in the affected hand region of area 3b that were obtained two weeks after the lesion. In New World monkeys, the representations of digits 1 to 5 in area 3b are arranged in a lateral to medial sequence, with the representations of distal hand located rostrally (digits) and the proximal hand (palm) located caudally (Sur et al., 1982). Interruption of more than 90% of the cervical dorsal column inputs is known to temporarily silence the neurons in hand cortex, and alter the somatotopic pattern that emerges over 4–6 weeks of recovery (Jain et al., 1997). In monkey SM-Rol (Fig. 4a), the DCL involved most of the cuneate fasciculus and spinal gray matter at C4 on the right side. The most medial region of the dorsal columns was spared. Comparison of the labeled terminal fields in the Cu nuclei of the intact and the deprived sides after the B-HRP injections into the matching parts of both hands suggested that approximately 9% of dorsal column afferents from below the lesion were spared from the lesion. In other words, 91% of the tactile inputs from the hand were disrupted. Two weeks after the DCL, neurons in nearly one third of the recording sites in the hand region of area 3b were unresponsive to touch on the hand, and 69.8% of the recording sites were only weakly responsive to touch on digits 1 to 5. The representations of the digits were roughly arranged in a somatotopic manner, while the representation of digit 3 was extra-large and the representations of digits 4 and 5 were too caudal in area 3b. Despite these abnormal features, we found the pattern of corticocuneate projections to be roughly similar to those of control monkeys (Fig. 5). The CTB injection in the deprived hand region of Cu labeled a large number of neurons in contralateral cortex (Fig. 5b). Most of the labeled neurons were located in the hand regions of areas 3a, 3b, 1 and 2, with slight expansions into the adjacent arm and face regions, and the forelimb region of M1. Note that the hand region of area 3b has a lower density of labeled neurons when compared to hand regions of other sensorimotor areas. Clusters of CTB-labeled neurons were also located in the PV/S2, PMv, and Cg regions. A few CTB-labeled neurons were found in other somatosensory areas along the lateral sulcus, PMd, SMA, and in the frontal cortex. We identified a small number of CTB-labeled neurons in the cortex ipsilateral to the injection (3.6% of the total labeled neurons in the two hemispheres; Fig. 5c, Table 2). The hand regions in areas 3a and 2 had the most labeled neurons, although the numbers are small. Few CTB-labeled neurons were observed in the areas 3b and 1, M1, the cortex of the lateral sulcus, PMd, SMA, and Cg in this case.

Figure 4.

The somatotopic maps of the hand region in area 3b after 2 weeks recovery from the incomplete dorsal column lesion (DCL) at cervical spinal segment 4 (C4) of SM-Rol (a; estimated 91% complete) and SM-Soo (b; estimated 91% complete). In both monkeys, the DCL involved most of the cuneate fasciculus but spared the most medial part of dorsal columns (top left on each panel). The dark gray shading in the transverse view of reconstructed spinal cord sections marks the lesion area. Neurons in parts of the area 3b hand cortex were unresponsive to touch on the hand, while some neurons responded weakly or well to touch on the hand in a roughly normal somatotopic pattern. CS, central sulcus; D1-D5, digits 1, 2, 3, 4, 5; LLP, lower lip; M, medial; R, rostral. Scale bar is 1 mm. The cortical map and transverse view of spinal cord injury for SM-Soo (b) are adapted from Liao et al., 2015.

Figure 5.

Distribution of labeled neurons in the flattened cortex after CTB was injected into the hand region of Cu of SM-Rol with a short-term, incomplete DCL (estimated 91% complete) at C4. (a) Photomicrograph showing the injection site in Cu. (b) The distribution of CTB-labeled neurons in the contralateral cortex resembles the normal pattern. The CTB-labeled neurons are distributed in the hand regions of areas 3b, 3a, 1 and 2, and PV/S2, and the forelimb region of M1. Neurons in other cortical areas including the PMd, PMv, SMA, PR, Ri, VS, and Cg are also labeled. (c) In the cortex ipsilateral to the Cu injection, some CTB-labeled neurons are distributed in the hand regions of areas 3a and 2, and fewer are in the M1, PMd, PMv, the lateral sulcus, and Cg. Note that the ipsilateral area 3b hand region has only 2 CTB-labeled neurons. Scale bar is 1 mm in (a), and 5 mm in (b–c). Other conventions as in Figure 1.

We observed similar results in monkey SM-Soo that also had been recovering from incomplete DCL (estimated 91% complete) at C4 for 2 weeks. Results from the standard mapping session revealed that nearly all neurons (93.5% of the recording sites) in the area 3b hand region were weakly or only very weakly responsive to touch on the hand (Fig. 4b). Only a few neurons in the expected representations of digits 1 and 2 responded well to touch on these digits. Again, the CTB injection in the deprived Cu labeled many neurons in the contralateral cortex (Fig. 6), with the majority in the hand-arm regions of areas 3a, 1 and 2, PV/S2, and M1, and other somatosensory areas in the lateral sulcus, PMv, PMd, SMA, and Cg. A smaller number of labeled neurons were found in the hand region of area 3b. Occasionally, CTB-labeled neurons appeared in the face regions in the somatosensory and motor cortex.

Figure 6.

Distribution of labeled neurons in the flattened cortex after CTB was injected into the hand region of Cu of SM-Soo with a short-term, nearly complete DCL (estimated 91% complete) at C4. (a) Photomicrograph showing the injection site in Cu. (b) The distribution of CTB-labeled neurons in the cortex contralateral to the Cu injection. Scale bar is 1 mm in (a), and 5 mm in (b). Other conventions as in Figure 1.

Complete DCLs at C4

We next examined the organization of corticocuneate connections in two monkeys with the short-term and nearly complete DCLs. In monkey SM-Rue (Fig. 7), the lesion involved the entire cuneate fasciculus on the right side at the C4 level, with expansion into the dorsal and intermediate zones of spinal gray matter. The ventral part of the dorsal columns and parts of spinal gray matter on the left side were also involved. After 2 weeks of recovery, we did not find any neurons in the hand regions of areas 3b and 1 responsive to touch on the hand, although strong neural responses to touch on the upper lip and chin were identified in the adjacent face region. Thus, the hand region was completely inactivated by the lesion. The brain was cut in the coronal plane to better reveal the laminar distribution of labeled neurons (Fig. 8), although the overall distribution of labeled neurons across cortical areas is less apparent in this cutting plane. The CTB-labeled neurons were found in the hand regions of somatosensory area 3b, 3a, 1/2, and the upper bank of the lateral sulcus including the PV/S2, PR, and Ri contralateral to the Cu injection. Motor cortex including M1, PMd, and rostral portion of posterior parietal cortex (PPCr) were also labeled. Some CTB-labeled neurons were in the SMA and Cg of the medial wall. A similar distribution of CTB-labeling was shown in the cortex ipsilateral to the injection but with a much smaller number (11.1% of the total labeled neurons in the two hemispheres). In order to determine the laminar distribution of these labeled neurons, we used the architectonical information revealed by the adjacent VGLUT2-processed sections to depict the location of layer 4 in the CTB processed sections. Labeled neurons were almost completely confined to the upper portion of the infragranular layer V, consistent with the findings of earlier studies in monkeys and cats (Cheema et al., 1985; Cheema et al., 1983). Only a small number of labeled neurons were located in the deeper portions of layer V.

Figure 7.

The somatotopic maps of the hand region in area 3b after 2 weeks recovery from the nearly complete DCL at C4 of SM-Rue (a; estimated 100% complete) and SM-LM (b; estimated 97% complete). (a) In SM-Rue, the DCL involved the entire cuneate fasciculus. Neurons in the area 3b hand cortex were unresponsive to touch on the hand. (b) In SM-LM, the DCL involved most of the cuneate fasciculus but spared the most dorsolateral region. Nearly all neurons in the area 3b hand cortex were unresponsive. Only a few neurons were weakly responsive to touch on the hand. Scale bar is 1 mm. Other conventions as in Figure 4. The cortical maps and transverse views of spinal cord injury for SMRue (a) and SM-LM (b) are adapted from Liao et al., 2015.

Figure 8.

Distribution of labeled cortical neurons in the coronally cut sections after CTB was injected into the hand region of Cu of SM-Rue with a short-term, nearly complete DCL (estimated 100% complete) at C4. (a) Photomicrograph showing the injection site in Cu. (b) The CTB-labeled neurons are mostly located in the hand regions of areas 3a, 3b 1 and 2, and PV/S2 of the contralateral hemisphere. Several motor areas including M1, PMd and the rostral portion of posterior parietal cortex (PPCr) are also labeled, and some CTB-labeled neurons are present in the Cg. Few CTB-labeled neurons are found in the cortex ipsilateral to the Cu injection. Note that nearly all the labeled neurons are located in the layer V. The dashed lines depict the center of layer IV that is revealed by the adjacent sections processed for vesicular glutamate transporter 2 (VGLUT2). Scale bar is 1 mm in (a). The numbers located left of the section drawings are serial section numbers. Other conventions as in Figure 1.

Monkey SM-LM had an estimated 97% complete DCL at C4 (Fig. 7b). Only a small part of the most dorsolateral part of the cuneate fasciculus was spared. The 2-week post-lesion mapping in the hand region of area 3b revealed that neurons in most of the recording sites did not respond to touch on the contralateral hand. Neurons weakly responsive to touch on digit 1 or tapping of the whole hand were found in few recording sites (10.3% of the recording sites). The locations of labeled neurons in the contralateral cortex after the Cu injection were plotted in the flattened cortical sections to reveal the overall distribution (Fig. 9). Large numbers of labeled neurons were found in the hand regions of areas 3a, 3b, 1 and 2 and PV/S2, and the forelimb region of M1. Note that the density of labeled neurons appeared to be lower in the hand region of area 3b. We also found clusters of labeled neurons in the expected locations of Ri, VS, PMd, and PMv, and few in the SMA, Cg, and frontal cortex.

Figure 9.

Distribution of labeled neurons in the flattened cortex after CTB was injected into the hand region of Cu of SM-LM with a short-term, nearly complete DCL (estimated 97% complete) at C4. (a) Photomicrograph showing the injection site in Cu. (b) The distribution of CTB-labeled neurons in the cortex contralateral to the Cu injection. The CTB-labeled neurons are mostly distributed in the hand-arm regions of area 3a, 1 and 2, and PV/S2. Note that fewer are present in the hand region of area 3b. Additional sensorimotor areas including the forelimb region of M1, and PMd, PMv, SMA, and Cg are also labeled. Scale bar is 1 mm in (a), and 5 mm in (b). Other conventions as in Figure 1.

In summary, the corticocuneate projection in monkeys with the short-term DCL, either incomplete or complete, closely resembled the patterns obtained from control monkeys, although the density of labeled neurons in the hand region of area 3b appeared to slightly decrease. Note that neurons in the affected hand region of area 3b were mostly inactivated or weakly responded to touch on the hand after 2 weeks of recovery.

Long-term recovery group

Incomplete DCLs at C4

To examine whether the corticocuneate connections are altered after longer recovery periods, CTB was injected into the deprived Cu at least 7 months after DCL (incomplete DCL, n = 2; nearly complete, n = 2). A unilateral DCL at the C4 level on the right side in monkey SM-D (Fig. 10a) extended dorsoventrally and involved most of the dorsal columns and the gray matter. The most medial and dorsolateral regions of the dorsal columns were spared, resulting in estimated 87% complete DCL. After 209 days of recovery, neurons throughout much of the affected hand region of areas 3b and 1 were responsive to touch on hand with modest to robust activities (76.1% of the recording sites). The representations of digits 1 to 5 were arranged in a lateromedial sequence, with the adjoining representation of palm located caudally. However, neurons responsive to touch on multiple digits or the entire hand were found at some sites, and responsiveness to touch on both hand and arm was detected at one recording site. The representations of face and arm were identified in the expected locations.

Figure 10.

The somatotopic maps of the hand region in area 3b after long-term recovery from the incomplete DCL at C4 of SM-D (a; estimated 87% complete; 209 days of recovery) and SM-P (b; estimated 76% complete; 245 days of recovery). In both monkeys, the DCL involved a large portion of the dorsal columns but spared the most dorsolateral and ventromedial regions. Nearly all neurons in the area 3b hand cortex were responsive to touch on the hand in a nearly normal somatotopic pattern. Scale bar is 1 mm. Other conventions as in Figure 4. The cortical maps and transverse views of spinal cord injury for SM-D (a) and SM-P (b) are adapted from Liao et al., 2018.

The Cu injection in SM-D produced a much more extensive distribution of labeled neurons in the cortex (Fig. 11) when compared to the results from control monkeys and monkeys with the short-term DCL (Figs. 1–3, 5, 6, 8, 9). Abundant CTB-labeled neurons were distributed in the hand regions of areas 3a, 1, and PV/S2, and the forelimb region of M1 in the contralateral cortex. In the somatosensory areas, many labeled neurons spread medially into their arm regions, and some extended laterally into the face regions. Moderate numbers of labeled neurons were in the hand and arm regions of areas 3b and 2, other somatosensory areas of the lateral sulcus, PMd, PMv, and Cg. Smaller numbers were in the SMA, cortex caudal to the area 2, the PPC, and frontal cortex. Additional labeled neurons were scattered across these areas, forming a continuous labeling pattern in the sensorimotor cortex rostral and caudal to the central sulcus, and in the cortex of the lateral sulcus. In the hemisphere ipsilateral to the Cu injection, small numbers of CTB-labeled neurons were present in the areas 3a and 2, M1, PV/S2, Ri, and Cg, but notably, we did not find labeled neurons in the hand regions of areas 3b and 1.

Figure 11.

Distribution of labeled cortical neurons in the flattened cortex after CTB was injected into the hand region of Cu of SM-D with a long-term, incomplete DCL (estimated 87% complete) at C4. (a) Photomicrograph showing the injection site in Cu. (b) The distribution of CTB-labeled neurons in the cortex contralateral to the Cu injection. The CTB-labeled neurons are predominately distributed in the hand regions of areas 3b, 3a, 1 and 2, and PV/S2, with extension into the arm and face regions. Other areas including the forelimb region of M1, and PMd, PMv, SMA, PR, Ri, VS, and Cg also have many CTB-labeled neurons. Note the labeling pattern is extensive. (c) In the cortex ipsilateral to the Cu injection, some CTB-labeled neurons are distributed in the hand-arm regions of areas 3a and M1, PV/S2, and Cg regions, and fewer are in the PMd and PMv. Note that area 3b hand region has no CTB-labeled neurons. Scale bar is 1 mm in (a), and 5 mm in (b–c). Other conventions as in Figure 1.

Monkey SM-P had an incomplete DCL (estimated 76% complete; Fig. 10b). After 245 days of recovery, the affected hand regions of areas 3b and 1 were widely reactivated to touch on hand with a roughly somatotopic organization (96.5% of the recording sites). After aligning the plotting of CTB-labeling with the adjacent NeuN sections that showed the layers in the coronal plane, we found that the labeled neurons were nearly completely confined to layer V of the somatosensory cortex including areas 3b, 3a, 1 and 2, and PV/S2, and M1 contralateral to the injection (Fig. 12). Clusters of CTB-labeled neurons were also found in the Ri and Cg, with fewer in PPCr. In the cortex ipsilateral to the injection, only a few labeled neurons were found in the layer V of areas 1 and 2, M1, and Cg. Only 1 labeled neuron was in area 3b.

Figure 12.

Distribution of labeled cortical neurons in the coronally cut sections after CTB was injected into the hand region of Cu of SM-P with a long-term, incomplete DCL (estimated 76% complete) at C4. (a) Photomicrograph showing the injection site in Cu. (b) The distribution of CTB-labeled neurons in the cortex. Note that nearly all the labeled neurons are located in the layer V. Scale bar is 1 mm in (a). Other conventions as in Figures 1 and 8.

Nearly complete DCLs at C4

Two monkeys (SM-W and SM-Rog) had nearly complete DCLs at C4 and long recovery times. Monkey SM-W had a DCL that involved the entire dorsal columns and spinal dorsal horn at C4 on the right side (Fig. 13a). Evaluation of the DCL extent from the B-HRP injections suggested a 100% complete DCL. After 251 days of recovery, area 3b neurons responsive to touch on arm and face were found in the expected locations. However, nearly half of the affected hand region of area 3b remained unresponsive to touch on the hand, while some neurons responded weakly to touch on the face or arm, or both. Occasionally, neurons responded weakly to touch on hand, but mostly with larger receptive fields that also involved the face and arm (8.5% of the recording sites). Thus, much of the hand region of area 3b was unresponsive to touch on the hand, and most of the neurons at responsive sites were somatotopically abnormal. Yet, neurons responsive to touch on the single digit 3 were found. Similar to the results obtained from monkeys with long-term and incomplete DCL at C4, the CTB injection in the deprived Cu labeled a great number of neurons in the contralateral cortex in an extensive pattern, to a lesser extent in the ipsilateral cortex (Fig. 14, Table 2). The majority of CTB-labeled neurons were located in the hand region of areas 3b, 1 and 2, PV/S2, with some spreading into the arm regions, and the forelimb region of M1. A small number of labeled neurons were found in the face regions of these areas. Only a few labeled neurons were present in the area 3a in this case, possibly because the injection missed most of the ECu. Many CTB-labeled neurons were also observed in the PR, Ri, and VS in the cortex of lateral sulcus, PMv, SMA, and Cg regions. In cortex ipsilateral to the injection, labeled neurons were scattered across cortical areas. Some CTB-labeled neurons were present in the hand-arm regions of areas 3a, 1 and 2, and M1. The somatosensory cortex in the lateral sulcus, PMd, PMv, SMA, and Cg were also labeled. Note that only two CTB-labeled neurons were found in the hand region of area 3b. Similar results were obtained from SM-Rog with a nearly complete DCL and 231 days postlesion recovery. We found weak responsiveness to touch on hand in 27.7% of affected hand cortex in area 3b. The Cu injection labeled a large number of neurons in the contralateral cortex, spreading across the sensorimotor cortex rostral and caudal to the central sulcus, the cortex along the lateral sulcus, and the Cg region (see details in Figs. 13b, 15). Some labeled neurons were identified in the ipsilateral cortex, mainly in the M1, areas 3a and 2, and the cortex of lateral sulcus.

Figure 13.

The somatotopic maps of the hand region in area 3b after long-term recovery from the incomplete DCL at C4 of SM-W (a; estimated 100% complete; 251 days of recovery) and SM-Rog (b; estimated 100% complete; 231 days of recovery). In both monkeys, the DCL involved the entire the dorsal columns. Nearly half or more of the area 3b hand cortex was unresponsive to touch on the hand, and many neurons responded weakly to touch on the arm and/or face. Neurons in few recording sites responded to touch on the hand. Scale bar is 1 mm. Other conventions as in Figure 4. The cortical maps and transverse views of spinal cord injury for SM-W (a) and SM-Rog (b) are adapted from Liao et al., 2018.

Figure 14.

Distribution of labeled cortical neurons in the flattened cortex after CTB was injected into the hand region of Cu of SM-W with a long-term, nearly complete DCL (estimated 100% complete) at C4. (a) Photomicrograph showing the injection site in Cu. (b) The distribution of CTB-labeled neurons in the cortex contralateral to the Cu injection. The CTB-labeled neurons are distributed in the hand and arm regions of areas 3b, 1 and 2, M1, and PV/S2, with a slight expansion into the face regions. The PMd, PMv, SMA, PR, Ri, VS and Cg are also labeled with many CTB-labeled neurons. Note that only a few neurons are labeled in the area 3a hand cortex. (c) The distribution of CTB-labeled neurons in the cortex ipsilateral to the Cu injection. Note only 1 labeled neuron is in the hand region of area 3b. Scale bar is 1 mm in (a), and 5 mm in (b-c). Other conventions as in Figure 1.

Figure 15.

Distribution of labeled cortical neurons in the flattened cortex after CTB was injected into the hand region of Cu of SM-Rog with a long-term, nearly complete DCL (estimated 100% complete) at C4. (a) Photomicrograph showing the injection site in Cu. (b) The distribution of CTB-labeled neurons in the cortex contralateral to the Cu injection. Note the labeling pattern is more extensive. (c) The distribution of CTB-labeled neurons in the cortex ipsilateral to the Cu injection. Scale bar is 1 mm in (a), and 5 mm in (b-c). Other conventions as in Figure 1.

Nearly complete DCLs at C5

Two monkeys (OM-St and OM-A) received the DCL at C5, which deprived most of the inputs from digits 2–5 while sparing many inputs from digit 1 and probably some from digit 2. The DCL in monkey OM-St was large (estimated 96% DCL complete) involving nearly the entire dorsal columns (cuneate and gracile fasciculi) and spinal dorsal horn on the left side (Fig. 16a). After 297 days, more than half of the hand region of area 3b did not respond to touch on hand, face or arm. However, some neurons responsive to touch on digits 1 and 2 (20.3% of recording sites) were randomly found mediolaterally throughout the hand cortex. This observation suggested that spared hand inputs activated both a few somatotopically appropriate sites in area 3b, but also other somatotopically non-matched sites over time. Occasionally, we found neurons weakly responsive to touch on the arm and both face and hand. The Cu injection ipsilateral to the lesion produced an impressively extensive distribution of labeled neurons in the contralateral cortex. Included cortex are the areas 3b, 3a, 1 and 2, M1, PMv, PMd, the cortex along the lateral sulcus, SMA, and Cg. Note that the CTB-labeled neurons were heavily distributed in the hand, arm, as well as the face regions in these areas (Fig. 17), possibly due to the slight spread of CTB injection halo into the spinotrigeminal nucleus in the brainstem, or the growth of face axon into the Cu. In addition to these densely labeled cortex, many labeled neurons were scattered around in the frontal, parietal, and temporal cortex. We also observed a much broader labeling in the cortex ipsilateral to the injection site in this monkey. Many CTB-labeled neurons were present in the M1 and area 3a, and fewer were in the area 3b, 1 and 2, and PMd, and PMv. Additional labeled neurons were seen in the cortex of the lateral sulcus, especially the VS area, Cg, and the frontal cortex. Note that few were in the hand region of area 3b, but more were in the face region.

Figure 16.

The somatotopic maps of the hand region in area 3b after long-term recovery from the incomplete DCL at cervical spinal segment 5 (C5) of OM-St (a; estimated 96% complete; 297 days of recovery) and OM-A (b; estimated 97% complete; 302 days of recovery). In both monkeys, the DCL involved nearly the entire dorsal columns. Over half of the area 3b hand cortex was unresponsive to touch on the hand. However, neurons responsive to touch on the digits 1, 2 or 3, or larger receptive fields in hand were identified in the lateromedial extent of hand cortex, suggesting an abnormal somatotopy. Other conventions as in Figure 4.

Figure 17.

Distribution of labeled cortical neurons in the flattened cortex after CTB was injected into the hand region of Cu of OM-St after a long-term recovery from a nearly complete DCL (estimated 96% complete) at C5. (a) Photomicrograph showing the injection site in Cu. (b) The distribution of CTB-labeled neurons in the cortex contralateral to the Cu injection. The CTB-labeled neurons are distributed in the hand, face and arm regions of areas 3b, 3a,1 and 2, M1, and PV/S2. PMd, PMv, SMA, PR, Ri, VS, Cg are also labeled with many CTB-labeled neurons. The labeling pattern is clearly extensive and widespread. (c) The distribution of CTB-labeled neurons in the cortex ipsilateral to the Cu injection. Note that CTB-labeled neurons are widely distributed across the cortical areas, but the area 3b hand region has only 5–6 labeled neurons. Scale bar is 1 mm in (a), and 5 mm in (b–c). Other conventions as in Figure 1.

Similar results were obtained from another monkey (OM-A) with an estimated 97% complete DCL at C5 and 302 days of recovery (Fig. 16). The hand region of area 3b was largely unresponsive, with a few neurons in scattered locations having receptive fields on digit 1, other parts of the hand, face or arm. The Cu injection labeled abundant neurons in the contralateral cortex, and smaller numbers of neurons in the ipsilateral cortex. The labeled neurons in both hemispheres had extensive and diffuse distributions (Fig. 18). Note, however, that fewer labeled neurons were in the regions of areas 3b and 1 that normally represent the digits.

Figure 18.

Distribution of labeled cortical neurons in the flattened cortex after CTB was injected into the hand region of Cu of OM-A after a long-term, nearly complete DCL (estimated 97% complete) at C4. (a) Photomicrograph showing the injection site in Cu. (b) The distribution of CTB-labeled neurons in the cortex contralateral to the Cu injection. (c) The distribution of CTB-labeled neurons in the cortex ipsilateral to the Cu injection. Note that the labeling pattern is more extensive in the two hemispheres. Scale bar is 1 mm in (a), and 5 mm in (b–c). Other conventions as in Figure 1.

In summary, after long recoveries from DCLs, the extent of cortical reactivation of area 3b hand cortex varied based on the lesion extent and level. In monkeys with extensive, nearly complete DCLs at C4, many neurons in the area 3b hand cortex remained unresponsive to touch on the hand, face or arm, but a few neurons became responsive to larger areas on the hand, face, arm, or multiple parts. Instead, most neurons throughout the area 3b hand cortex responded well to touch on the hand with a nearly normal somatotopic pattern in monkeys with incomplete DCLs at C4. In monkeys that had large, nearly complete DCLs at C5 with intentionally spared inputs from digit 1 and often digit 2, most of area 3b remained silent to touch on hand, but neurons at a few scattered locations were responsive to touch on digits 1 or 2. However, the distributions of neurons projecting to the deprived Cu in all monkeys with the longer recoveries were notably denser and more extensive across the hand, face, and arm regions of areas 3b, 3a, 1 and 2, PV/S2, and M1 in the contralateral cortex when compared to the results obtained from normal monkeys or monkeys with short-term recoveries. This increase in density and range of labeled neurons was even more pronounced in monkeys with DCLs at C5, which allowed inputs from digits 1 and perhaps 2 to continue to project to the deprived Cu during the recovery periods. More extensive distributions of labeled neurons were also present in the ipsilateral cortex but in a significantly smaller number. Note that only a few labeled neurons were found in the hand region of area 3b of the ipsilateral cortex.

Potentiation of the corticocuneate projection and cortical reactivation

Results from the present cases indicate that DCLs may alter the number of neurons projecting from the area 3b hand region to the deprived Cu over longer recovery times. Data from 3 normal monkeys, 4 monkeys with the short-term DCL, and 6 monkeys with the long-term DCL are presented in Table 3. The monkeys with the DCL were further categorized based on the lesion level (C4 or C5) and extent (incomplete or nearly complete). Given the possible variations in tracer injection size and transportation efficiency across cases, the percentage of labeled neurons in the area 3b in the contralateral cortex in each monkey may not clearly reflect the change of projections after the injury. Therefore, we used the ratio of the DCL projection to the normal projection for corticocuneate projections from the area 3b hand cortex to the total from the contralateral hemisphere in each DCL case (Fig. 19a). Our results indicate that the proportion of labeled neurons in area 3b hand cortex compared to the normal projection increased by 1.4× in monkeys with the short-term and incomplete DCL at C4, but remained similar in monkeys with the short-term and nearly complete DCL (1.0×). However, we observed increases of the ratio in monkeys with the long-term DCL at C4 (incomplete: 1.2×; nearly complete: 1.3×). The proportion nevertheless decreased in monkeys with the long-term and nearly complete DCL at C5. Due to the low number of cases in each group, we did not test for statistically significant differences in the proportions between groups (Fig. 19a). Instead, we focused on the relationship of the corticocuneate projection to cortical reactivation. The contribution of the corticocuneate projection from area 3b hand cortex to reactivate the area 3b hand cortex was analyzed in 10 monkeys with the DCL. We divided the hand cortex in area 3b based on the neural responsiveness to touch on the hand, on the body areas outside the hand (face, arm, or larger area involving face and arm), or unresponsive. The percentage of cortical maps with the neural receptive fields in the hand was then related to the proportion of labeled neurons in the area 3b hand cortex. The results suggest that the proportion of labeled neurons in the area 3b hand cortex has no clear correlation with the percent of recording sites with responses to touch on the hand (Pearson’s r = 0.2086, P = 0.563; Fig. 19b). Instead, the percent of functional reactivation in the affected hand region of area 3b was found to be significantly correlated with the extent of DCL (Pearson’s r = −0.8593, P = 0.0014; Fig. 19c). In summary, our results suggest that the extent of functional recovery in the area 3b hand cortex after the DCL is less related to the number of feedback projections from the affected hand region of area 3b to the Cu, but more related to the lesion extent in the spinal cord.

Figure 19.

The relationships of corticocuneate projection to the DCL extent, recovery times, and the cortical reactivation in the area 3b hand cortex. (a) Bar graphs show the average ratios of corticocuneate projections from the area 3b hand region for DCL versus the average normal projection, grouped by lesion extent and recovery period. These ratios of labeled neurons in the area 3b hand cortex contralateral to the Cu injection are shown for each monkey as black dots within the bar graph. (b) Pearson’s analysis indicated that the ratio of labeled neurons in the area 3b hand cortex for DCL versus the normal average is not correlated with the percentage of recording sites in the area 3b hand cortex that are responsive to touch on the hand. (c) The extent of cortical reactivation to touch on the hand in area 3b hand cortex, nevertheless, has a negative correlation with the lesion extent. The gray zone in (b-c) depicts the 95% confidence intervals, and the black line represents the linear trend line. R² = coefficient of determination from linear regression.

DISCUSSION

Our goals were to describe the normal organization of cortical projections from two hemispheres to the Cu, and to determine if this projection pattern is changed by lesions of the dorsal column inputs from the hand that activate neurons of the Cu (Cliffer and Willis, 1994; Rustioni et al., 1979; Willis and Coggeshall, 1991). Nearly complete lesions immediately and completely deactivate the digit and hand representation in area 3b of contralateral cortex (Jain et al., 1997; Jain et al., 2008; Kaas et al., 2008; Liao et al., 2015). Over time, the few preserved inputs reactivate portions of the hand cortex in area 3b. Partial lesions are followed by large and more complete reactivations, and the area 3b reactivation is matched by the reactivation of higher-level somatosensory areas 1, S2 and PV (Qi et al., 2018; Yang et al., 2014). Hand use in retrieving food recovers with cortical reactivation (Qi et al., 2013), but locomotion is not hampered by the DCL. Our results indicate that cortical neurons projecting to Cu are extensive in normal monkeys, and not restricted to hand representations in sensory and motor areas. After short-term deactivations of the Cu and hand cortex, most connections were preserved, but the density of labeled neurons appeared to be lower in area 3b hand cortex. After long recovery times and partial reactivations, labeled cortical neurons were more widespread (Fig. 20).

Figure 20.

Summary diagram showing the organizations of corticocuneate projection in normal monkeys (blue), and monkeys with the short-term (orange) and long-term (red) DCLs. The cuneate nucleus normally receives cortical inputs from the hand regions of sensorimotor cortex predominantly in the contralateral and fewer in the ipsilateral hemispheres. This cortical descending influence remains largely similar in New World monkeys with the short recoveries from the DCL, but is greatly increased after the long recoveries.

The projection pattern of normal monkeys

The normal patterns of corticocuneate projection were highly similar in the two owl monkeys and the single squirrel monkey. The projections from contralateral area 3b (S1) were largely from neurons in the hand representation where they would be activated by stimulating the hand, and could have receptive fields that overlapped, or were near the receptive fields of the Cu neurons they targeted. The labeled neurons were densely distributed in the portion of area 3b between the hand-face border septum and the lateral end of the shallow central sulcus that mark the lateral and medial borders of the digit and palm representations, but were nearly absent from the more medial portion of area 3b representing the wrist, arm, trunk, leg and foot. However, numbers of labeled neurons were found lateral to the hand representation in cortex that represents the face, but not in even more lateral cortex representing the teeth and tongue.

Area 1 hand region was also populated with labeled neurons. However, the distribution of labeled neurons included representations of the arm and trunk, as well as some of the face. Thus, many of the labeled area 1 neurons could terminate on Cu neurons with somatotopically matched or nearby receptive fields, but perhaps half or more would not. The area 2 representation is defined here as a band of cortex along the caudal border of area 1 and of the same width as area 1. Area 2 has not been mapped in any small New World monkeys (Padberg et al., 2005). However, area 2 has been mapped in Old World macaques (Padberg et al., 2019; Pons et al., 1985) and large New World cebus monkeys (Padberg et al., 2007), so that an existence of area 2 with a somatotopy in parallel to that in area 1 is likely in all monkeys. Much of area 2 in mapped monkeys is devoted to the hand, as is the caudally adjoining PPC (Krubitzer and Kaas, 1992). Thus, some or most of the population of labeled neurons in the area 2 region could have receptive fields on the hand, but others would not.

Other populations of labeled neurons appeared to be by location in the regions of the S2, PV, and VS, all of which are somatotopically arranged, and have neurons with larger receptive fields than neurons in area 3b (Coq et al., 2004; Cusick et al., 1989; Disbrow et al., 2003; Disbrow et al., 2000; Friedman et al., 1980; Krubitzer et al., 1995; Qi et al., 2002; Wu and Kaas, 2003). The parts of S2 and PV nearest area 3b and along the presumptive area 1 border that represent the face are relatively free of labeled neurons. Labeled neurons are mostly on the upper bank of the lateral sulcus where the hand is represented. Thus, some or most of the labeled neurons in the PV/S2 region, and even in VS, could have receptive fields on the hand that overlap those in the Cu neurons. Less is known about the PR and Ri regions, except that they have connections with somatosensory areas such as S2 and PV (Disbrow et al., 2003; Krubitzer and Kaas, 1990; Qi et al., 2002). The receptive fields of the labeled neurons scattered in these areas of the lateral sulcus are thus uncertain, but unlikely to all be somatotopically matched with neurons in the Cu neurons. Area 3a contains a somatotopic map of proprioceptive afferents that parallels the map of tactile afferents in area 3b (Huffman and Krubitzer, 2001; Kaas et al., 1979; Merzenich et al., 1978; Nelson et al., 1980).

The M1, PMd, and PMv have representations that are crudely somatotopic, but more directly reflect classes of motor actions than somatotopy (Godschalk et al., 1995; Graziano et al., 2002; Mitz and Wise, 1987; Stepniewska et al., 1993). However, many or most of the labeled neurons in area 3a, M1, and premotor cortex likely relate to the functional use of the hand and arm, and thus could usefully modulate the flow of sensory information from the Cu to cortex. The significance of the labeled neurons in other motor related regions, including the SMA and Cg is uncertain, but perhaps related to motor activities involving the hand and arm. Interestingly, the projection neurons from the ipsilateral cortex are few, perhaps constituting 10–20% of the total, and none would have matching sensory receptive fields of Cu neurons.

Previous descriptions of normal cortical projections to Cu in primates

Overall, our results in squirrel monkeys and owl monkeys are similar to those described previously in Old World monkeys (Bentivoglio and Rustioni, 1986; Catsman-Berrevoets and Kuypers, 1976; Cheema et al., 1985; Rustioni and Weinberg, 1989), although some discrepancies are noted. While earlier studies in macaque monkeys reported that tracer injections in Cu labeled large numbers of neurons in the areas 1 and 2, and many in areas 3a and 3b (Catsman-Berrevoets and Kuypers, 1976; Cheema et al., 1985; Weisberg and Rustioni, 1977), we did not find such sharp contrast (see Table 3). These reported differences may reflect where injections were placed within the longitudinal structure of Cu. Cheema et al. (1985) found that areas 1 and 2 project to the entire length of Cu with the densest distribution in the rostral sector, and the projection from the area 3b mainly targeted the middle “core” region of Cu. Area 3a had a relatively sparse projection that mainly targeted the ventral part of Cu and the medullary reticular formation ventral to the Cu (Jones and Wise, 1977). Most of the earlier studies injected larger amounts (0.25 – 70 μl) of horseradish peroxidase (HRP) or HRP-conjugated tracers that spread throughout the rostrocaudal extent of Cu and involved adjacent medullary nuclei (Catsman-Berrevoets and Kuypers, 1976; Cheema et al., 1985; Weisberg and Rustioni, 1977), whereas we utilized electrophysiological recording to assure that the small amount of CTB (0.1–0.2 μl) injected targeted the representation of digits (the “core”) in Cu. Thus, differences in the injection strategies likely led to most of the differences in labeling patterns between previous studies and present results. Of course, we did not fully avoid the spread of the injection core along the rostrocaudal extent of the core sector of Cu, which could contribute to the differences in results between monkeys, in addition to the possibilities of the individual differences.

Another discrepancy is that almost all previous studies reported that the sensorimotor cortex and DCN have somatotopically congruent connections (Bentivoglio and Rustioni, 1986; Cheema et al., 1985; Coulter and Jones, 1977), while our findings included labeled neurons in the hand, arm, and face regions in these areas after CTB was injected into the electrophysiologically-identified hand representation of Cu. Some labeling of neurons in the arm regions is expected since the representation of arm is immediately next to the representation of digits within Cu (Xu and Wall, 1999b), and the injection core could extend into the arm region. In contrast, reasons for the labeling of neurons in the face regions of areas 3b and 1 are uncertain, since the injection cores did not involve the adjacent trigeminal nucleus based on the microscopic observations.

Proposed functions of corticocuneate projections

The proposed functions of corticocuneate projections derive from the premise that they are at least roughly somatotopic, and are largely based on previous studies in cats and rats (Aguilar et al., 2003; Canedo, 1997; Canedo and Aguilar, 2000; Castellanos et al., 2007; Malmierca and Nunez, 1998; Marino et al., 2000; Marino et al., 1999; Palmeri et al., 1999). Thus, somatotopically matched cortical inputs from these major somatosensory and motor areas could facilitate the responsiveness of target neurons in the Cu, while those with nearby receptive fields could have suppressive effects on Cu neurons. These thereby contribute to noise suppression and enhance the spatial and temporal precision of somatosensory stimuli. The cellular mechanisms include the direct activation of the Cu neurons that have the overlapping receptive fields through the NMDA and non-NMDA receptors, and disinhibition of GABAergic influence on these Cu neurons via serial activations of glycinergic- GABAergic interneurons, and suppression of Cu neurons that have non-overlapping receptive fields through intrinsic GABAergic interneurons (Aguilar et al., 2003). Malmierca and Nunez (2004) also reported similar modulatory mechanisms of S1 projections to neurons in the gracile nucleus (Gr) that had somatotopically matched and non-matched receptive fields. Furthermore, the feedback projections from S1 promote the spike synchronization between the spatially distributed Gr neurons that have overlapping receptive fields, thus enhancing the temporal resolution of responses to peripheral inputs (Castellanos et al., 2007; Malmierca et al., 2009; Nunez and Malmierca, 2007).

The roles of projection neurons in the representations of especially the face, but also arm and trunk, are less clear. These connections in normal monkeys may reflect some spread of the tracer outside the Cu, and this should be checked by placing injections in cortex with anterograde tracers. The functional roles of the labeled neurons in SMA, insular, and Cg regions, as well as neurons in ipsilateral cortex, have not been investigated, but these regions are involved in sensorimotor functions, and may contribute to overall suppression, and thus noise reduction. The functional role of projection from the ipsilateral hemisphere to the Cu is unclear. But if from the hand and arm regions, they could contribute to large, suppressive receptive field surrounds, as they do in area 3b, where neurons with small receptive fields on individual digits have large suppressive surrounds that not only include other digits on the same hand, but the ipsilateral hand as well (Reed et al., 2011; Tommerdahl et al., 2006).

It is possible that neurons with no somatosensory receptive fields provide part of the cortical projections to the Cu. What is clear, is that a large proportion of the cortical projection to the Cu involves neurons with mismatched receptive fields, and the functions of such connections remain uncertain. These are likely to have suppressive effects.

The organization of corticocuneate projections after short-term recoveries from DCLs

We studied the distributions of corticocuneate projections in 4 monkeys with Cu injections immediately before a DCL, followed by a 2-week period for neural transport and labeling. After this 2-week period, the Cu and higher stations would be inactive with complete or nearly complete lesions, and some retraction of axon arbors of corticocuneate projection might be expected. Alternatively, the growth of nearby axons into the silent zone could occur. Most likely, such changes so early in the recovery process would be limited or absent. In the cases with large lesions, the majority of the affected hand region in area 3b remained silent to touch on the hand (SM-Rue & SM-LM, Fig. 7). Occasionally, we found neurons weakly responsive to touch on the dorsal side of digit 1, or on large areas of the glabrous and dorsal hand. The distribution of projection neurons in the cortex is better demonstrated by case SM-LM in which the brain was cut parallel to the brain surface. The labeled neurons were primarily located in the hand regions of areas 3a, 3b, 1 and 2, PV/S2, and motor cortex including the M1 and premotor cortex, with a small number of labeled neurons in the SMA and Cg regions. The overall pattern is similar to the results from the control monkeys, although the number of labeled neurons in the hand region of area 3b was slightly less than expected. Similar results were obtained in monkeys with less complete lesions, SM-Rol (Fig. 5) and SM-Soo (Fig. 6). Many neurons in the hand region of area 3b responded weakly to touch on the hand in the somatotopic pattern. The Cu injection again labeled the neurons at the cortical level in a pattern that was indistinguishable from the control cases and the cases with the short-term, nearly-complete DCL.

Overall, in the 2-week period after DCL, the distributions of labeled corticocuneate neurons appeared to be normal or nearly normal. Those with somatotopically congruent inputs would not have normal patterns of activation, and would be likely nearly unresponsive when the lesion is extensive. Therefore, the descending cortical projections do not change or change very little over a 2-week period as a result of nearly complete deactivations of Cu and cortical neurons.

The organization of corticocuneate projection after long-term recoveries from DCL

After long recovery periods of more than 7 months, monkeys with the DCL had variable extents of cortical reactivations in the hand cortex of primary somatosensory cortex area 3b, which were largely determined by the lesion extents and levels in the spinal cord. In monkeys with incomplete DCLs at C4, sparing parts of the inputs from the hand, most area 3b hand neurons regained the responsiveness to touch on the hand, and the somatotopy was nearly normal (SM-D & SM-P, Fig. 10). In monkeys with the nearly complete DCL at C5 that spared the digit 1 input but deprived most of the inputs from digits 2 to 5 (OM-St & OM-A, Fig. 16), an abnormal reactivation occurred in the area 3b hand cortex, with the representation of digit 1 identified in the unexpected locations. Even cases with nearly complete DCLs at C4 (SM-W & SM-Rog, Fig. 13) had some reactivation to touch on the hand, although some neurons that became responsive to touch on the arm and/or face. The somatotopically matched reactivations to touch on the hand are largely driven by preserved direct and indirect secondary inputs to the Cu (Liao et al., 2015; Liao et al., 2018; Qi et al., 2016), while the non-matched reactivations to touch on the face and arm suggest that sensory inputs to the hand region of Cu may include axons from the face (Jain 2000, Liao 2016) and from the arm as a result of axon growth. The growth or potentiation of connections intrinsic to the area 3b hand region may also have a role mediating the reactivation process (Liao et al., 2016).

Along with the more extensive and variable cortical reactivations, the Cu injections in monkeys with the long-term DCLs produced drastically more widespread distributions of labeled neurons in both cortical hemispheres when compared to the control monkeys and monkeys with the short-term DCL, suggesting large-scale patterns of axon growth from cortical neurons into the Cu occur during the weeks to months of post-lesion recovery. Thus, proportionately lower cortical inputs would be from neurons with overlapping receptive fields, and a facilitative role.

Plasticity during the recovery period after the DCL

Several factors could result in anatomical alterations of the corticocuneate pathway over time, although it is not directly injured by the DCL. First, the environment of the target area, the Cu, is greatly changed after the DCL, including a sudden silencing of neural activity that were normally driven by the lost spinal cord inputs, imbalanced synaptic interactions between neurons, and a slow, chronic atrophy of deactivated neural populations within the nucleus (Darian-Smith et al., 2010). Second, the subsequent relay stations at the thalamus, primary somatosensory cortex area 3b, and other somatosensory and motor areas, especially their hand regions, likely undergo anatomical and neurochemical modifications due to the loss of normal inputs (e.g., Wang et al., 2016; Freund et al., 2011). In our previous cases, we found that DCLs greatly reduce the expression of the VGLUT2 within the first two post-lesion weeks, while returning to normal levels over seven months of recovery (unpublished data). Similarly, decreasing the activity in the Cu in squirrel monkeys by crushing the median nerve decreased the level of mature glutamate receptor subunit expression when evaluated one week later (Sarin et al., 2012). Such results suggest that a great reduction of neural activities in the Cu results in a short-term down regulation of molecular mechanisms for maintaining normal activity. During this early deprivation, changes in GABA receptor subunits also occur (Mowery et al., 2011), suggesting a period of reduced or altered cellular inhibition. These influences on the corticocuneate pathway could be dynamic and continually modulated in response to activity, behavior, and skill acquisition over weeks to months, and contribute to the sprouting or retractions during the recovery process.

The more widespread distribution of corticocuneate projection neurons is more apparent in monkeys with more spared dorsal column inputs from the hand to the Cu and months of recovery, suggesting that the existence of surviving driving inputs to the Cu influences the recruitment of more widespread and non-specific cortical inputs to the Cu. Previous studies in rodents (e.g., Hill et al., 2001) and macaque monkeys (e.g., Darian-Smith et al., 2014) suggested similar time windows for axonal growth or withdrawals during weeks to months of recovery from the spinal cord injury. The axonal arbors of these descending projections in the Cu may also expand to innervate more Cu neurons in response to the DCL. This speculation is based on the previous findings that the corticospinal projections from M1 and areas 3b and 1 contralateral to the lesion sprouted significantly and bilaterally beyond normal range in macaque monkeys at 4–5 months after DCLs (Darian-Smith et al., 2014; Fisher et al., 2020; Fisher et al., 2018). In addition, approximately 40–60% of these corticospinal projection neurons send collateral axons to the brainstem Cu in macaque monkeys (Bentivoglio and Rustioni, 1986). Thus, injuries that caused these neurons to expand their axon terminal fields in the spinal cord may simultaneously trigger the sprouting of their collateral branches in the Cu.

While the increased cortical inputs to the deprived Cu could be beneficial in restoring the neural activity in Cu over time, this descending pathway may have more of a suppressive, modulatory influence on Cu neurons during the recovery. The anatomical pattern of corticocuneate projection does not suggest a predominance of connections between neurons of matching or overlapping receptive fields in the cortex and Cu, and the alterations in the connection pattern during recovery do not reflect the extent of cortical reactivation in the affected hand region of area 3b after the injury. In monkeys with the DCL that spared some dorsal column inputs from the hand, potentiation of these inputs to Cu may be facilitated by matching cortical inputs to the Cu. However, in monkeys with a nearly complete DCL, cortical descending inputs to the deprived Cu increased to include more neurons with somatotopically non-matched inputs from the face or arm, possibly introducing abnormal patterns of neural reactivation and perceptual confusion. The role of new inputs from the somatotopically non-matched cortex or nonsensorimotor cortex during the recovery period is yet unclear.

Finally, in many of our cases, the lesion extended somewhat beyond the dorsal columns into the spinal gray matter and sometimes the ventral white matter to possibly damage local segmental circuits (e.g., Willis and Coggeshall, 1991; Wu et al., 2019) and motor connections (e.g., Darian-Smith, 2009). While these larger lesions could affect the functional recovery after injury, we have no clear evidence on the effects of DCLs that go beyond those of the loss of primary inputs to the Cu.

Conclusions

The results indicate that the cuneate nucleus (Cu) normally receives descending projections primarily from the hand representations in somatosensory and motor areas, with a small portion of cortical inputs from the ipsilateral cortex. Other inputs are from somatotopically mismatched parts of sensory and motor cortex, as well as from cortex with other functions. In monkeys with the short-term recoveries from DCLs, and most of the affected area 3b hand cortex unresponsive or weakly responsive to touch on hand, the organization of corticocuneate projections closely resembles the normal pattern. In monkeys with DCLs and longer post-lesion recoveries, an expansion of the cortical labeling pattern was apparent, especially when the lesion spared more inputs from the hand to the Cu. The expanded descending pathway would likely alter the signaling transmission in the Cu depending on the available peripheral inputs to the Cu. The spared primary afferents and the second-order pathway are the major sources that drive the functional recovery after the DCL.