Robert W. Rhoades's research while affiliated with Medical University of Ohio at Toledo and other places

In neonatal rodents, elevated levels of cortical serotonin (5-HT) blur the normally segmented vibrissae-related pattern of thalamocortical afferents (TCAs) in the posteromedial barrel subfield (PMBSF) of primary somatosensory cortex. We employed 5-HT immunocytochemistry or anterograde transport of 1'1'-dioctadecyl-3,3,3',3' tetramethyl-indocarbocyanin (Di-I) to label TCA arbors to study the effects of 5-HT manipulations on space occupied by TCAs within the PMBSF and the total area labeled. In rats treated to increase cortical 5-HT from birth to postnatal day (P) 6, the percentage of PMBSF area occupied by terminal labeling was significantly higher from that in controls (79.0% versus 23.7%, P < 0.05) for the highest levels of cortical 5-HT and was raised, although not significantly, for lower levels of 5-HT. The TCA coverage was significantly correlated with treatment dose. In animals exposed to a selective 5-HT1B agonist, 5-nonyloxytryptamine, or elevated endogenous 5-HT, the total areas of TCA aggregates in the PMBSF and those in visual cortex were similar to the controls. These results suggest that TCAs have a graded response to increasingly higher 5-HT concentrations. The lack of TCA expansion beyond normal cortical areas further implies that 5-HT-induced axon outgrowth is restricted at cortical boundaries.

In adult rats that sustained forelimb amputation on the day of birth, >30% of multiunit recording sites in the forelimb-stump representation of primary somatosensory cortex (SI) also respond to cutaneous hindlimb stimulation when cortical GABA(A+B) receptors are blocked (GRB). This study examined whether hindlimb receptive fields could also be revealed in forelimb-stump sites by reducing one known source of excitatory input to SI GABAergic neurons, the contralateral SI cortex. Corpus callosum projection neurons connect homotopic SI regions, making excitatory contacts onto pyramidal cells and interneurons. Thus in addition to providing monosynaptic excitation in SI, callosal fibers can produce disynaptic inhibition through excitatory synapses with inhibitory interneurons. Based on the latter of these connections, we hypothesized that inactivating the contralateral (intact) SI forelimb region would "unmask" normally suppressed hindlimb responses by reducing the activity of SI GABAergic neurons. The SI forelimb-stump representation was first mapped under normal conditions and then during GRB to identify stump/hindlimb responsive sites. After GRB had dissipated, the contralateral (intact) SI forelimb region was mapped and reversibly inactivated with injections of 4% lidocaine, and selected forelimb-stump sites were retested. Contralateral SI inactivation revealed hindlimb responses in approximately 60% of sites that were stump/hindlimb responsive during GRB. These findings indicate that activity in the contralateral SI contributes to the suppression of reorganized hindlimb receptive fields in neonatally amputated rats.

In adult rats that sustained forelimb amputation on the day of birth, there are numerous multi-unit recording sites in the forelimb-stump representation of primary somatosensory cortex (SI) that also respond to cutaneous stimulation of the hindlimb when cortical receptors for GABA are blocked. These normally suppressed hindlimb inputs originate in the SI hindlimb representation and synapse in the dysgranular cortex before exciting SI forelimb-stump neurons. In our previous studies, GABA (A + B) receptor blockade was achieved by topically applying a bicuculline methiodide/saclofen solution (BMI/SAC) to the cortical surface. This treatment blocks receptors throughout SI and does not allow determination of where along the above circuit the GABA-mediated suppression of hindlimb information occurs. In this study, focal injections of BMI/SAC were delivered to three distinct cortical regions that are involved in the hindlimb-to-forelimb-stump pathway. Blocking GABA receptors in the SI hindlimb representation and in the dysgranular cortex was largely ineffective in revealing hindlimb inputs ( approximately 10% of hindlimb inputs were revealed in both cases). In contrast, when the blockade was targeted at forelimb-stump recording sites, >80% of hindlimb inputs were revealed. Thus GABAergic interneurons within the forelimb-stump representation suppress the expression of reorganized hindlimb inputs to the region. A circuit model incorporating these and previous observations is presented and discussed.

Studies of sensory pathways in several species indicate that the extent and form of reorganization resulting from deafferentation early in life vs. adulthood are not the same. The reasons for such differences are not well understood. To gain further insight into age-dependent mechanisms of reorganization, this study compared the consequences of neonatal vs. adult forelimb amputation in rats at multiple levels of the sensory pathway, including primary somatosensory cortex, brainstem, and dorsal root ganglia. At the cortical level, the average area of the functional forelimb-stump representation from rats amputated as adults was significantly smaller (P < 0.05) than that of neonatally amputated rats (4.3 +/- 1.3 mm(2) vs. 6.6 +/- 1.5 mm(2), respectively). At the brainstem level, neonatally amputated rat cuneate neurons possessed the following responsivities: 20% stump responsive, 40% responsive to both stump and hindlimb, 30% responsive to another body region, and 10% unresponsive. In contrast, cuneate neurons of adult amputated rats were 70% stump responsive, 2% responsive to both stump and hindlimb, and 30% unresponsive. A significantly (P < 0.001) greater percentage of the C(6)-C(8) dorsal root ganglia neurons of adult amputated rats were unresponsive to peripheral stimulation vs. neurons from neonatally amputated rats (48% vs. 16%, respectively). These results indicate that the reorganization that occurs in response to forelimb amputation at birth vs. adulthood is distinctly different at each of these levels of the dorsal column-medial lemniscal pathway. Possible mechanisms to account for these differences are considered.

Rats that sustain forelimb removal on postnatal day (P) 0 exhibit numerous multi-unit recording sites in the forelimb-stump representation of primary somatosensory cortex (SI) that also respond to hindlimb stimulation when cortical GABAA+B receptors are blocked. Most of these hindlimb inputs originate in the medial SI hindlimb representation. Although many forelimb-stump sites in these animals respond to hindlimb stimulation, very few respond to stimulation of the face (vibrissae or lower jaw), which is represented in SI just lateral to the forelimb. The lateral to medial development of SI may influence the capacity of hindlimb (but not face) inputs to "invade" the forelimb-stump region in neonatal amputees. The SI forelimb-stump was mapped in adult (>60 days) rats that had sustained amputation on embryonic day (E) 16, on P0, or during adulthood. GABA receptors were blocked and subsequent mapping revealed increases in nonstump inputs in E16 and P0 amputees: fetal amputees exhibited forelimb-stump sites responsive to face (34%), hindlimb (10%), and both (22%); neonatal amputees exhibited 10% face, 39% hindlimb, and 5% both; adult amputees exhibited 10% face, 5% hindlimb, and 0% both, with approximately 80% stump-only sites. These results indicate age-dependent differences in receptive-field reorganization of the forelimb-stump representation, which may reflect the spatiotemporal development of SI. Results from cobalt chloride inactivation of the SI vibrissae region and electrolesioning of the dysgranular cortex suggest that normally suppressed vibrissae inputs to the SI forelimb-stump area originate in the SI vibrissae region and synapse in the dysgranular cortex.

Elevating cortical serotonin (5-HT) in rats with clorgyline, a monoamine oxidase A (MAO(A)) inhibitor, from postnatal day (P-0) to P-6 delays the organization of thalamocortical afferent fibers into a vibrissae-related pattern in the somatosensory cortex (S-I). Despite continued elevation of cortical 5-HT through P-8, the thalamocortical fibers do form, albeit with some delay, a characteristic vibrissae pattern of barrels in layer IV of S-I by P-8. The growth-associated protein, GAP-43, is transiently expressed in developing S-I cortex of normal rats in a vibrissae related pattern until P-7. After P-7, GAP-43 expression is reduced in the barrel centers and increased in the septa. The present study evaluated the effect of elevated 5-HT levels on the distribution of GAP-43 immunoreactivity in S-I. We employed 5-HT immunocytochemistry and 1,1'-dioctadecyl-3,3,3",3'-tetramethylindocarbocyanine perchlorate (DiI) labeling of thalamic radiations to confirm a 'barrelless' phenotype in P-6 clorgyline-treated animals and a recovered barrel pattern in treated animals allowed to survive until P-8 and P-10. GAP-43 immunocytochemistry was used to evaluate the cortical distribution of this protein in similarly treated littermates. Continuous inhibition of MAO(A) from P-0 to P-6 resulted in a corresponding loss of the GAP-43 vibrissae-related pattern at P-6. Despite continued elevation of cortical 5-HT until P-8 and P-10, the characteristic vibrissae-complementary pattern of GAP-43 emerged with expression concentrated in the septa and rows. GAP-43 vibrissae-related thalamocortical axon pattern never appeared in the clorgyline-treated animals. Thus, while elevated 5-HT delays development of a vibrissae-related pattern of thalamocortical afferents, it does not appear to alter the time when a GAP-43 vibrissae-related complementary pattern emerges.

Previous studies have shown that intracortical projections in layer IV of the vibrissae representation of primary somatosensory cortex (S-I) are arrayed in a pattern complementary to that of thalamocortical axons (TCAs). Elevation of cortical serotonin (5-HT) in rats during the first postnatal week results in a transient disruption of the vibrissae-related pattern of TCAs and layer IV neurons in S-I. The present study examines the influence of elevated cortical 5-HT levels and the attendant loss of vibrissae-related TCA clusters on the organization of S-I intracortical connections. Cortical 5-HT was elevated in neonatal rats via chronic injections of clorgyline from birth until P-6. Animals were euthanized on P-6 or allowed to survive an additional 4 days without further clorgyline treatment. Distributions of TCAs and intracortical axons were assessed via application of 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (Di-I) and 4-(4-(dihexadecylamino)styryl)-N-methylpyridinium iodide (Di-A) to the thalamic radiations and directly into the cortical barrelfield, respectively. Chronic administration of clorgyline resulted in a loss of the vibrissae-related organization of TCAs in layer IV of S-I. There was also a loss of the complementary pattern of intracortical projections in layer IV of this region. Discontinuation of clorgyline treatment resulted in a return of the vibrissae-related pattern of TCAs as well as the complementary pattern of intracortical projections. These results are consistent with the conclusion that the normal organization of intracortical projections in this region of S-I depends on the presence of the orderly array of TCAs.

Elevating cortical serotonin (5-HT) in rats from postnatal day (P-) 0 to P-6 by administering the monoamine oxidase (MAO(A)) inhibitor, clorgyline, produces a dose-dependent spectrum of effects on rat somatosensory organization, ranging from enlarged with indistinct septa to a complete lack of vibrissae-related patterns. However, if clorgyline treatment is stopped on P-6, a qualitatively and quantitatively normal vibrissae-related pattern of thalamocortical afferents appears in somatosensory cortex (S-I) on P-10. We employed high performance liquid chromatography (HPLC), infraorbital nerve (ION) transection, N-methyl-D-aspartate (NMDA) receptor blockade, 1,1'-dioctadecyl-3,3,3"3'-tetramethylindocarbocyanine perchlorate (DiI) labeling of thalamic afferents, and CO histochemistry to determine whether peripheral nerve input and/or cortical NMDA receptor activity were required for the recovery of vibrissae-related patterns in clorgyline-treated animals. Clorgyline administration from P-0 to P-6 produced a 1589.4+/-53.3% increase in cortical 5-HT over control animals on P-6 and a 268.8+/-6.3% elevation over controls at P-10. Postnatal day 6 pups had significantly altered vibrissae-related patterns in S-I following 6 days of clorgyline treatment but by P-10, the characteristic vibrissae-related patterns were restored. Neither transection of the ION nor application of the NMDA antagonist, DL-2-amino-5-phosphonovaleric acid (APV), to the cortices of P-6 pups that were treated with clorgyline from birth had any significant effect on the recovery of the vibrissae-related patterns by P-10. These results indicate that neither peripheral nerve input nor cortical NMDA receptor activity are necessary for the restoration of cortical vibrissae-related patterns in rats that have sustained transient elevations of 5-HT.

Previous studies from this laboratory showed that sprouting of serotoninergic (5-HT) axons in the hamster's superior colliculus (SC), induced by a single subcutaneous injection of 5,7-dihydroxytryptamine (5,7-DHT) at birth (postnatal day 0 [P-0]), resulted in an increased terminal distribution of the uncrossed retinocollicular projection that was not associated with any changes in the number or distribution of ipsilaterally projecting retinal ganglion cells. The present study was undertaken to determine what effect this manipulation had on the terminal arbors of such axons. Retinocollicular axons of normal and 5,7-DHT-treated animals were anterogradely labeled with small intraretinal injections of the lipophilic dye 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) on P-16. After tissue processing on P-19, single retinocollicular axon arbors were reconstructed by using confocal microscopy. Quantitative analysis indicated that arbors from 5,7-DHT-treated hamsters had significantly greater total fiber lengths, areas, and volumes than those from normal animals. There were no differences between axons from the two groups in number of branch points, distribution of relative branch lengths, and numbers of bouton-like swellings. These results support the hypothesis that increased SC concentrations of 5-HT alter development of the uncrossed retinocollicular pathway such that a greater territory is covered by individual terminal arbors but that the number of synaptic contacts per arbor remains constant. This may explain, at least in part, the abnormally widespread distribution of the aggregate ipsilateral projection.

Neonatal transection of, or blockade of axoplasmic transport in, the infraorbital nerve [ION, the trigeminal (V) branch that supplies the mystacial vibrissae follicles] results in a loss of all central patterns corresponding to the vibrissae follicles in the brainstem, thalamus and cortex except for those of the central terminal arbors of ION primary afferents that survive this lesion. Both of these manipulations also result in a rapid and dramatic upregulation of at least two peptides, galanin and neuropeptide Y, in surviving vibrissae-related primary afferents. Galanin is of particular interest, because this peptide has effects on neuronal activity and growth, both factors which may be involved in the disappearance of central vibrissae-related patterns in rats that have sustained neonatal ION transection or axoplasmic transport blockade. The present study used antisense technology to determine whether the upregulation of galanin in the central terminals of ION primary afferents is necessary for the loss of central vibrissae-related patterns in rats. Newborn rats had their left ION transected or axoplasmic transport in this nerve blocked by application of a vinblastine-impregnated implant, and at the same time received an injection of commercially synthesized phosphorothioate oligodeoxynucleotide sequences (15-20 bases) directly into the V ganglion in order to block galanin upregulation. These injections effectively prevented the upregulation of this peptide which is normally associated with ION transection or axoplasmic transport blockade. Preventing galanin upregulation, however, did not prevent or attenuate the loss of central vibrissae-related patterns in the brainstem or cortex normally observed following ION transection or axoplasmic transport blockade in this nerve. These results are thus consistent with the conclusion that the upregulation of galanin in the central terminals of V primary afferents, observed after damage to or attenuation of axoplasmic transport in the ION, is not necessary for the reorganization that results in a disappearance of central vibrissae-related patterns in the V neuraxis.