Somatic activation of thalamic neurons transplanted into lesioned somatosensory thalamus

BrainResearch, 478 (1989)356-3.60 Elsevier

356 BRE 23319

Somatic activation of thalamic neurons transplanted into lesioned somatosensory thalamus Sharon L. Julianol, Isabelle Dusart 2 and Marc Peschanski 2 Department of Anatomy, USUHS, Bethesda, MD 20814 (U.S.A.) and 21NSERM, UI61, Paris(France)

(Accepted 11 October 1988) Key words: Ventrobasal complex; Homotypicgrafting; Fetal neuron; Cytochromeoxidase; Acetylcholinesterase; 2-Deoxyglucose;Metabolicactivity

Homotypicfetal neurons were transplanted into previouslylesioned ventrobasal complex of rats. After 1-3 months of survivalthe animals receivedinjectionsof 2-deoxy-[]4C]glucoseto reveal metabolicactivityof the transplanted cellsin response to somaticstimuli. These experimentsindicated that stimulus-evokedactivityin the transplants of animals receivinga somatic stimuluswas significantly greater than in the transplants of animals that were not stimulated. Control studies using cell counts, cytochro~aeoxidase and acetylcholinesterase histochemistryestablished that the differences in activityvalues were not due to the number of survivingcells or the metabolic health of the individualgrafts.

A complete loss of neurons results from injections of kainic acid into the ventrobasal complex of rats 12. The neuronal depletion in this somatosensory thalamic nucleus is paralleled by deficits in the behavioral response to somatic stimulation l°. Despite these dramatic alterations, the lemniscai afferent fibers, which are deprived of their targets, remain present and viable in the lesioned area for months ml3. Transplants of homotypic fetal thalamic neurons into the neuron-depleted area cause host lemniscal fibers to form contacts with the transplanted cells that appear similar to normal synapses 15. The present study explores the potential of these host-graft connections to convey inputs evoked by peripheral somatic stimulation, using the 2-deoxyglucose (2-DG) technique. We present evidence using [14C]2-DG, cytochrome oxidase and acetyicholinesterase histochemistry, that grafted neurons are functionally and anatomically integrated into the host CNS. These results suggest that recovery might be induced in the CNS after lesions of sensory systems through the use of homotypic transplantation. Fourteen rats were used in this study. Each animal

received a stereotaxic injection of kainic acid (KA) into the right ventrobasal thalamus (VB). One month later, the rats were reanesthetized and received a graft of dissociated fetal thalamic tissue (gestational age 15 days) into the lesioned area following the methods described in ref. 1. For 8 animals, the donor dams were injected with [3H]thymidine on 2 different occasions (gestational days 12 and 14) and thus received transplants which were identifiable (Fig. 1). The animals survived for 1-3 months after the transplantation. At this point, a 2-DG experiment was performed; half of the animals received a somatic stimulus, the remaining animals received no somatic stimulation. During the 2-DG experiment, each rat was unanesthetized, lightly restrained and intravenously injected with 2-deoxy-o-[14C]glucose (10 /~Ci/100 g). Half of the animals received an intermittent vertical displacement stimulus delivered at 3 Hz bilaterally to the ventral surface of the hindpaw; after 2-DG injection the stimulation continued for 45 min. The remaining animals received no stimulus. Both the stimulus and non-stimulus groups were given an overdose of pentobarbital Na and perfused with sa-

Correspondence: S.L. Juliano, Department of Anatomy, USUHS, 4301Jones Bridge Road, Bethesda, MD 20814, U.S.A.

0006-8993/89/$03.50t~) 1989Elsevier Science PublishersB.V. (BiomedicalDivision)

357

%

Fig. 1. Section taken through a 1-month-old graft in which the

donor dam was injected with [3H]thymidine. Clusters of cells labeled autoradiographically with thymidine are indicated with arrow heads. The section is counterstained with thionin for identification of Nissl substance. Numerous gliai cells can be seen surrounding the graft. line followed by 4% buffered paraformaldehyde. The brains were quickly removed and frozen. Later, brain blocks were cut on a cryostat; adjacent sections were saved for 2-DG autoradiography, acetylcholinesterase (ACHE) or cytochrome oxidase (CO) histochemistry. The sections which produced the autoradicgraphs were stained for Nissl substance. Quantification of the metabolic label was achieved using a video-based image processing system which converted the optical density valaes i~ito ~4C-concentrations. The ~4C-values were expressed as percent above background, where background was considered to be the white matter of the corpus callosum. The transplants were identified in the Nissl-stained sections using criteria established in previous studies ~4. The distinguishing characteristics of the transplants included their isolation from the host neurons by a layer of glial cells and the assembly of the grafted neuronal somata into clusters. Three transplants were found not to be localized in the lesioned VB area. In both the stimulated and unstimulated ani-

Fig. 2. Digitized 2-deoxyglucose autoradiographs taken from 2 different animals with fetal thalamic cells transplanted one month earlier. A is from an animal which received a somatic stimulus, B is from an unstimulated animal. The transplants are indicated with open arrows (x 18). The 2-DG activity in the graft in A is 55% above background; the 2-DG activity in B is 33% above background. mals the pattern of metabolic activity was distributed in a fluctuating manner (Fig. 2). This uneven distribution of label was coincident with the clusters of neurons identified on the Nissl-stained sections. In the unstimulated animals, the metabolic activity averaged 53% above background in the left (intact) VB and 31% above background in the graft (Table I). No differences were observed in the transplants allowed to survive for different amounts of time. In the stimulated animals, regions of the left (intact) VB which receive inputs from the hindlimb averaged 70%

358 TABLE 1 Activity values for 2-DG and CO activity in normal VB and transplants in the opposite hemispheres

Values are expressed as percent above background. Survival time (months)

2-DG

CO

Intact VB/unstimulated GAK 2 GAK 4 GAK 6 GAK 10

3 2 1 3

56% 52% 53% 51%

58% 43% 53% 43%

Intact VB/stimulated GAK 1 GAK 3 GAK 5 GAK 13

3 2 1 2

74% 75% 71% 61%

48% 49% 60% 45%

Grafted VB/unstimulated GAK 2 GAK 4 GAK 6 GAK 10

3 2 1 3

33% 28% 37% 27%

63% 42% 54% 42%

Grafted VB/stimulated GAK 1 GAK 3 GAK 5 GAK 13

3 2 1 2

55% .54% 55% 47%

47% 51% 56% 43%

above background for the optical density values (Table I, Fig. 2). In the same stimulated rats, the level of metabolic activity was 53% above background in the transplants on the opposite side; these values are significantly different from those found in the

transplants of the unstimulated animals (P < 0.001, paired t-test). The 2-DG label in the transplants did not appear to be topographically organized, or to occur in any particular region of the graft. The results of the 2-DG experiment therefore indicate that a stimulus-evoked activity occurred in the transplants, since the ~4C-concentrations found in animals that received a somatic stimulation were significantly greater than those found in the transplants of the unstimulated rats. Control experiments verified that the increased metabolic label resulted from increased activity in the grafted neurons. (i) To eliminate a potential bias toward low or high activity values introduce d by differences in cellular density of the transplants in the unstimulated versus the stimulated groups, cells were counted using the Nisslstained sections. Differences in the cell density between the two populations of transplants were not statistically significant. (ii) The pattern and level of CO activity was evaluated as an index of the chronic functional state of the grafts. In contrast to 2-DG activity, the C O activity is not sensitive to phasic stimuiaticm (Fig. 3a,b). The CO activity was quantified with the same image analysis system used for the 2D G analysis. The CO optical density values were expressed as a percent above a standard value obtained in each section; in this series the corpus callosum was used as the standard background value. The CO activity also occurred i~i clusters related to the clustered pattern of implanted neurons. The density and distribution of the C O activity, however, was comparable

Fig. 3. Sections demonstrating transplants stained for cytochrome oxidase (CO) (a,b), and acetylcholinesterase (ACHE) (c) activity in transplants which survived for one (a,b) or two (c) months (x28). The section in a is taken from a transplant in an animal that was not stimulated during the 2-DG experiment; the section in b is from an animal that was stimulated during the 2-DG study. The CO activity in the transplant shown in a is 54% above background and 56% above background in the transplant shown in b.

359 in the two populations of transplants (Table I). (iii) AChE staining was used to compare the level of maturation in the grafts since the AChE activity pattern distinguishes stages of thalamic development m~. In VB, A C h E levels are relatively weak in the adult, but transiently robust during maturation (the AChE stains heavily until 19 days post-natal). The pattern of staining was comparable in the stimulated and unstimulated rats, however it was also paradoxical, since the A C h E stained densely in all the animals, even though many of the transplants were in situ for 3 months. This observation suggests that the maturational level of the total population of rats was the same, although different from normal. The AChE distribution also occurred in clusters related to cell bodies (Fig. 3c). The experiments described above provide support for our contention that peripheral somatic stimulation produced metabolic activity increases in the neurons of the VB transplants. An alternative rationale for such stimulus-evoked increases might be explained by the demonstration that in some situations, 2-DG consumption is greater in fiber terminals than in cell bodies 9a6. We feel that in these experiments this possibility is not likely for two reasons, First, in the animals where the grafts were not well localized in the lesioned VB, neither the transplants nor the region of the lesion (which presumably contains ingrowing afferent fibers) contain above-background 2-DG activity. Second, in the animals with appropriately placed transplants, the lesioned regions surrounding the grafts, also containing ingrowing affer-

ents, do not demonstrate above background metabolic label (Fig. 2). Although a number of studies demonstrate that grafted neurons cause functional recoveries and develop connections with the host 2-s.7, the results presented here demonstrate an in vivo activation of transplanted neurons by host fibers belonging to a specific 'point-to-point' system. This result is similar to that obtained with Purkinje cells grafted into Purkinje cell degeneration mutant mice 17. In this model, the implanted Purkinje cells become progressively reinvested by the host fibers and a highly specific 'point-to-point' pattern of synapse formation develops. Electrophysiological recordings from the same preparation demonstrate that the transplanted Purkinje cells are activated by stimulation to the climbing fibers 6. The demonstration that grafted thalamic neurons are activated by stimulation of specific sensory afterents may provide part of the physiological basis for a behavioral recovery of somatosensory function. Before such a behavioral recovery can be certain, a functional reconnection between the grafted thalamic neurons and somatosensory cortical neurons must be verified. Other studies have shown that when homotypic grafts are implanted into an excitotoxically lesioned striatum, the previously deafferented target zones in the pallidum were functionally reinnervated by the grafted striatal neurons 8. A similar funct, ional reconnection must be clearly demonstrated in the thalamocortical somatosensory system.

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