Neural transplants in patients with Huntington's disease undergo disease-like neuronal degeneration.

The clinical evaluation of neural transplantation as a potential treatment for Huntington's disease (HD) was initiated in an attempt to replace lost neurons and improve patient outcomes. Two of 3 patients with HD reported here, who underwent neural transplantation containing striatal anlagen in the striatum a decade earlier, have demonstrated marginal and transient clinical benefits. Their brains were evaluated immunohistochemically and with electron microscopy for markers of projection neurons and interneurons, inflammatory cells, abnormal huntingtin protein, and host-derived connectivity. Surviving grafts were identified bilaterally in 2 of the subjects and displayed classic striatal projection neurons and interneurons. Genetic markers of HD were not expressed within the graft. Here we report in patients with HD that (i) graft survival is attenuated long-term; (ii) grafts undergo disease-like neuronal degeneration with a preferential loss of projection neurons in comparison to interneurons; (iii) immunologically unrelated cells degenerate more rapidly than the patient's neurons, particularly the projection neuron subtype; (iv) graft survival is attenuated in the caudate in comparison to the putamen in HD; (v) glutamatergic cortical neurons project to transplanted striatal neurons; and (vi) microglial inflammatory changes in the grafts specifically target the neuronal components of the grafts. These results, when combined, raise uncertainty about this potential therapeutic approach for the treatment of HD. However, these observations provide new opportunities to investigate the underlying mechanisms involved in HD, as well as to explore additional therapeutic paradigms.

Testis-derived Sertoli cells have a trophic effect on dopamine neurons and alleviate hemiparkinsonism in rats.

Neural tissue transplantation has become an alternative treatment for Parkinson's disease (PD) and other neurodegenerative disorders. The clinical use of neural grafts as a source of dopamine for Parkinson's disease patients, although beneficial, is associated with logistical and ethical issues. Thus, alternative graft sources have been explored including polymer-encapsulated cells and nonneural cells (that is, adrenal chromaffin cells) or genetically modified cells that secrete dopamine and/or trophic factors. Although progress has been made, no current alternative graft source has ideal characteristics for transplantation. Emerging evidence suggests the importance of trophic factors in enhancing survival and regeneration of intrinsic dopaminergic neurons. It would be desirable to transplant cells that are readily available, immunologically accepted by the central nervous system and capable of producing dopamine and/or trophic factors. Sertoli cells have been shown to secrete CD-95 ligand and regulatory proteins, as well as trophic, tropic, and immunosuppressive factors that provide the testis, in part, with its "immunoprivileged" status. The present study demonstrated that transplantation of rat testis-derived Sertoli cells into adult rat brains ameliorated behavioral deficits in rats with 6-hydroxydopamine-induced hemiparkinsonism. This was associated with enhanced tyrosine hydroxylase (TH) immunoreactivity in the striatum in the area around the transplanted Sertoli cells. Furthermore, in vitro experiments demonstrated enhanced dopaminergic neuronal survival and outgrowth when embryonic neurons were cultured with medium in which rat Sertoli cells had been grown. Transplantation of Sertoli cells may provide a useful alternative treatment for PD and other neurodegenerative disorders.

A comparison of dopaminergic cells from the human NTera2/D1 cell line transplanted into the hemiparkinsonian rat.

Human NT cells derived from the NTera2/D1 cell line express a dopaminergic phenotype making them an attractive vehicle to supply dopamine to the depleted striatum of the Parkinsonian patient. In vitro, hNT neurons express tyrosine hydroxylase (TH), depending on the length of time they are exposed to retinoic acid. This study compared two populations of hNT neurons that exhibit a high yield of TH+ cells, MI-hNT and DA-hNT. The MI-hNT and DA-hNT neurons were intrastriatally transplanted into the 6-OHDA hemiparkinsonian rat. Amelioration in rotational behavior was measured and immunohistochemistry was performed to identify surviving hNT and TH+ hNT neurons. Results indicated that both MI-hNT and DA-hNT neurons can survive in the striatum, however, neither maintained their dopaminergic phenotype in vivo. Other strategies used in conjunction with hNT cell replacement are likely needed to enhance and maintain the dopamine expression in the grafted cells.

Testis-derived Sertoli cells survive and provide localized immunoprotection for xenografts in rat brain.

Transplantation of neural tissue into the mammalian central nervous system has become an alternative treatment for neurodegenerative disorders such as Parkinson's disease. Logistical and ethical problems in the clinical use of human fetal neural grafts as a source of dopamine for Parkinson's disease patients has hastened a search for successful ways to use animal dopaminergic cells for human transplantation. The present study demonstrates that transplanted testis-derived Sertoli cells into adult rat brains survive. Furthermore, when cotransplanted with bovine adrenal chromaffin cells (xenograft), Sertoli cells produce localized immunoprotection, suppress microglial response and allow the bovine cells to survive in the rat brain without continuous systemic immunosuppressive drugs. These novel features support Sertoli cells as a viable graft source for facilitating the use of xenotransplantation for Parkinson's disease and suggest their use as facilitators, (i.e., localized immunosuppression) for cell transplantation in general.

Sertoli cells induce systemic donor-specific tolerance in xenogenic transplantation model.

Cell therapy is a potentially powerful tool in the treatment of many grave disorders including leukemia, immune deficiencies, autoimmune diseases, and diabetes. However, finding matched donors is challenging and recipients may suffer from the severe complications of systemic immune suppression. Sertoli cells, when cotransplanted with both allo- and xenograft tissues, promote graft acceptance in the absence of systemic immunosuppression. How Sertoli cells do this is not, as yet, clearly defined. We have examined the ability of Sertoli cells to produce systemic immune tolerance. For this purpose, Sertoli cells were injected into an otherwise normal C57/BL6 mouse host via the lateral tail vein. No other immunosuppressive protocols were applied. Six to 8 weeks posttransplantation, blood was collected for analysis of cytokine levels. Tolerance to donor cells was determined by mixed lymphocytic culture, and production of T-cell-dependent antibody was determined by an in vitro anti-sheep red blood cell plaque-forming assay. Results showed a marked modulation of immune cytokines in the transplanted mouse host and donor-specific transplantation tolerance was achieved. Tolerant mouse lymphocytes maintained a competent humoral antibody response. Additionally, C57/BL6 mice transplanted with rat Sertoli cells tolerated rat skin grafts significantly longer than control non-Sertoli cell transplanted mice. We conclude that systemic administration of rat Sertoli cells across xenogenic barrier induces transplantation tolerance without altering systemic immune competence. These data suggest that Sertoli cells may be used as a novel and potentially powerful tool in cell transplantation therapy.

Survival of rat or mouse ventral mesencephalon neurons after cotransplantation with rat sertoli cells in the mouse striatum.

Transplanting cells across species (xenotransplantation) for the treatment of Parkinson's disease has been considered an option to alleviate ethical concerns and shortage of tissues. However, using this approach leads to decreased cell survival; the xenografted cells are often rejected. Sertoli cells (SCs) are testis-derived cells that provide immunological protection to developing germ cells and can enhance survival of both allografted and xenografted cells. It is not clear whether these cells will maintain their immunosuppressive support of cografted cells if they are transplanted across species. In this study, we investigated the immune modulatory capacity of SCs and the feasibility of xenografting these cells alone or with allografted and xenografted neural tissue. Transplanting xenografts of rat SCs into the mouse striatum with either rat or mouse ventral mesencephalon prevented astrocytic infiltration of the graft site, although all transplants showed activated microglia within the core of the graft. Surviving tyrosine hydroxylase-positive neurons were observed in all conditions, but the size of the grafts was small at best. SCs were found at 1 and 2 weeks posttransplant. However, few SCs were found at 2 months posttransplant. Further investigation is under way to characterize the immune capabilities of SCs in a xenogeneic environment.

Behavioral alterations in Lewis rats following two-day continuous 3-nitropropionic acid administration.

The mitochondrial toxin, 3-nitropropionic acid (3-NP), produces motor dysfunction and striatal atrophy in rats. However, rat strain and method of administration may contribute to variability in the deficits caused by 3-NP toxicity. To evaluate this, changes in nocturnal spontaneous locomotor activity from chronic administration of 3-NP using an osmotic mini pump, were examined in the Lewis rats. Lewis rats were treated with 3-NP or saline for 2 days and behavior was tested daily for a 15 day period. Animals receiving 3-NP displayed significantly less spontaneous activity than animals in the saline group. 3-NP treated animals also weighed significantly less when compared to saline treated animals. These results demonstrate that even though there were no significant alterations in overt anatomical pathology, even short-term exposure to 3-NP produced significant effects. This short-term administration may present a potential paradigm for examination of sub-threshold neurotoxicity.

Neural transplantation of human neuroteratocarcinoma (hNT) neurons into ischemic rats. A quantitative dose-response analysis of cell survival and behavioral recovery.

Transplantation of fetal neuronal tissue has been used successfully to ameliorate symptoms of neurodegenerative disease in animals and humans. This technique has recently been extended as an experimental treatment for ischemic brain damage. However, due to ethical issues with the use of fetal cells for the treatment of any human disease, there has been a concerted effort to find alternative graft sources for neural transplantation. The human neuroteratocarcinoma neuron cell is derived from an embryonal teratocarcinoma cell line that can be differentiated into post-mitotic neurons. Neural transplantation of human neuroteratocarcinoma neurons has recently been shown to produce behavioral amelioration of symptoms in rats with ischemia-induced injury. The present study was undertaken to: (i) determine the minimum effective number of transplanted human neuroteratocarcinoma neurons required for amelioration of ischemia-induced behavioral dysfunction; and (ii) quantify the survival of human neuroteratocarcinoma neurons in vivo. Transplants of 0, 5, 10, 20, 40, 80 or 160 x 10(3) human neuroteratocarcinoma neurons were made into rats that sustained ischemic damage. Animals that received 40, 80 or 160 x 10(3) human neuroteratocarcinoma neurons demonstrated a dose-dependent improvement in performance of both the passive avoidance and elevated body swing tests. At the conclusion of behavioral testing, human neuroteratocarcinoma neurons were identified in paraffin sections with human neural cell adhesion molecule MOC-1 and human neurofilament antibodies. Transplants of 80 or 160 x 10(3) human neuroteratocarcinoma neurons demonstrated a 12-15% survival of human neuroteratocarcinoma neurons in the graft, while transplants of 40 x 10(3) human neuroteratocarcinoma neurons demonstrated a 5% survival. Transplantation of human neuroteratocarcinoma neurons ameliorated behavioral deficits produced by ischemic damage. The human neuroteratocarcinoma neuron, additionally, showed greater survival than that reported for fetal cells when transplanted into the brain. Therefore, this readily available cell may prove to be an excellent candidate for the treatment of ischemic damage in human patients.

Transplantation of human fetal striatal tissue in Huntington's disease: rationale for clinical studies.

Huntington's disease is a fatal neurological disorder characterized by chorea and deterioration in cognitive and neuropsychiatric function. Primary pathological changes are found in the striatum, where GABAergic neurons undergo degenerative changes. Local interneurons are relatively spared. Here, we describe the rationale for clinical trials of fetal striatal tissue transplantation for the treatment of Huntington's disease. Specifically, the reasons for utilizing tissue derived from the far lateral aspect of the lateral ventricular eminence as a source of striatal tissue will be discussed.

Formation of Sertoli cell-enriched tissue constructs utilizing simulated microgravity technology.

Cell transplantation therapy for diabetes and Parkinson's disease offers hope for long-term alleviation of symptoms. However, successful protocols remain elusive due to obstacles, including rejection and lack of tropic support for the graft. To enhance engraftment, testis-derived postmitotic Sertoli cells have been cotransplanted with islets in the diabetic rat (Db) and neurons in the Parkinsonian rat (PD). Sertoli cell tropic, regulatory, and nutritive factors that nourish and stimulate germ cells also support isolated neurons and islets in vitro. Likewise, immunosuppressive properties of Sertoli cells, extant in the testis, are expressed by extratesticular Sertoli cells evidenced by allo- and xenograft immunoprotection of grafts in both the CNS (in the PD model) and the periphery (in the Db model). On this basis, we have created Sertoli islet cell aggregates (SICA) and Sertoli neuron aggregated cells (SNAC) using simulated microgravity culture technology developed by NASA. Isolated rat and pig Sertoli cells were cocultured with neonatal pig islets (SICA) and with immortalized N-Terra-2 (NT2) neurons (SNAC) in the HARV biochamber. Formed aggregates were assayed for desirable functional and structural characteristics. Cell viability in SICA and SNAC exceeded 90% and FasL immunopositive Sertoli cells were present in both. Sertoli cells did not interfere with insulin secretion by SICA and promoted differentiation of NT2 cells to the dopaminergic hNT cell type in SNAC. Addition of Matrigel resulted in structural reorganization of the aggregates and enhanced insulin secretion. We conclude that SICA, SNAC, and Matrigel-induced islet- and neuron-filled "Sertoli cell biochambers" are suitable for long-term transplantation treatment of Db and PD.

Viability and survival of hNT neurons determine degree of functional recovery in grafted ischemic rats.

We recently reported behavioral improvements following intrastriatal transplantation of cryopreserved cultured human neuroteratocarcinoma-derived cells (hNT neurons) in rats with cerebral ischemia induced by occlusion of the middle cerebral artery. In the present study, the viability and survival of hNT neurons were evaluated immediately prior to the transplantation surgery and at 3 months post-transplantation in ischemic rats. Cryopreserved hNT neurons were routinely thawed, and trypan blue exclusion viability counts revealed 52-95% viable hNT neurons before transplantation. Monthly behavioral tests, starting at 1 month and extending to 3 months post-transplantation, revealed that ischemic animals that were intrastriatally transplanted with hNT neurons (approximately 40000) and treated with an immunosuppressive drug displayed normalization of asymmetrical motor behavior compared with ischemic animals that received medium alone. Within-subject comparisons of cell viability and subsequent behavioral changes revealed that a high cell viability just prior to transplantation surgery correlated highly with a robust and sustained functional improvement in the transplant recipient. Furthermore, histological analysis of grafted brains revealed a positive correlation between number of surviving hNT neurons and degree of functional recovery. In concert with similar reports on fetal tissue transplantation, we conclude that high cell viability is an important criterion for successful transplantation of cryopreserved neurons derived from cell lines to enhance graft-induced functional effects.

Bilateral fetal striatal grafts in the 3-nitropropionic acid-induced hypoactive model of Huntington's disease.

We investigated the 3-nitropropionic acid (3-NP)-induced hypoactive model of Huntington's disease (HD) to demonstrate whether fetal tissue transplantation can ameliorate behavioral deficits associated with a more advanced stage of HD. Twelve-week-old Sprague-Dawley rats were introduced to the 3-NP dosing regimen (10 mg/kg, i.p., once every 4 days for 28 consecutive days), and were then tested for general spontaneous locomotor activity in the Digiscan locomotor apparatus. All rats displayed significant hypoactivity compared to their pre-3-NP injection locomotor activity. Randomly selected rats then received bilateral intrastriatal solid grafts of fetal striatal (lateral ganglionic eminence, LGE) tissues from embryonic day 14 rat fetuses. Approximately 1/3 of each LGE in hibernation medium was infused into each lesioned host striatum. In control rats, medium alone was infused intrastriatally. A 3-mo posttransplant maturation period was allowed prior to locomotor activity testing. Animals receiving fetal LGE grafts exhibited a significant increase in locomotor activity compared to their post-3-NP injection activity or to the controls' posttransplant activity. Surviving striatal grafts were noted in functionally recovered animals. This observation supports the use of fetal striatal transplants to correct the akinetic stage of HD. To the best of our knowledge, this is the first study that has investigated the effects of fetal striatal transplantation in a hypoactive model of HD.

A unique cytoplasmic marker for extratesticular Sertoli cells.

In the absence of a definitive cell marker for testis-derived Sertoli cells, their identification in cell culture or in Sertoli cell-facilitated cell transplantation protocols is difficult and limits the creditable evaluation of experimental results. However, the production by prepubertal Sertoli cells of Mullerian inhibiting substance (MIS) presents the possibility of specifically identifying extratesticular Sertoli cells as well as Sertoli cells in situ, by the immunodection of this unique glycoprotein. This study was designed to determine if isolated rat Sertoli cells could be identified by routine immunocytochemistry utilizing an antibody raised against MIS. Sertoli cells immunostained for MIS included Sertoli cells in situ and freshly isolated, cultured and cocultured Sertoli cells, and Sertoli cells structurally integrated with NT2 cells in simulated microgravity. Detection of MIS was also determined by Western blot analysis.

Post-thaw viability and functionality of cryopreserved rat fetal brain cells cocultured with Sertoli cells.

Testis-derived Sertoli cells have been used to create an immune "privileged" site outside of the testis to facilitate cell transplantation protocols for diabetes and neurodegenerative diseases. In addition to secreting immunoprotective factors, Sertoli cells also secrete growth and trophic factors that appear to enhance the posttransplantation viability of isolated cells and, likewise, the postthaw viability of isolated, cryopreserved cells. It would be beneficial if Sertoli cells could be cryopreserved with the transplantable cell type without deleterious effects on the cells. This report describes a protocol for the cocryopreservation of rat Sertoli cells with rat ventral mesencephalic neurons, neurons from the lateral and medial ganglionic eminences and the hNT neuron cell line, and reports on the effects of Sertoli cells on the the postthaw viability of these neurons. Results of trypan blue exclusion analysis indicated that the presence of Sertoli cells did not deleteriously effect cryopreserved neurons and may improve their postthaw recoverability and viability in general. Specifically, results of the tyrosine hydroxylase immunostaining showed that Sertoli cells significantly enhance the postthaw viability of ventral mesencephalic dopaminergic cells in vitro.

Trophic effect of porcine Sertoli cells on rat and human ventral mesencephalic cells and hNT neurons in vitro.

The poor survival of embryonic dopaminergic (DA) neurons transplanted into patients with Parkinson's disease (PD) has encouraged researchers to search for new methods to affect the short- as well as long-term survival of these neurons after transplantation. In several previous rodent studies Sertoli cells increased survival of islet cells and chromaffin cells when cotransplanted in vivo. The aims of this study were to investigate whether porcine Sertoli cells had a positive effect on the survival and maturation of rat and human DA neurons, and whether the Sertoli cells had an effect on differentiation of neurons derived from a human teratocarcinoma cell line (hNT neurons). A significant increase of tyrosine hydroxylase (TH)-positive neurons of both rat and human ventral mesencephalic tissue was found when cocultured with Sertoli cells. Furthermore, there was a significantly increased soma size and neurite outgrowth of neurons in the coculture treated group. The Sertoli cell and hNT coculture also revealed an increased number of TH-positive cells. These results demonstrate that the wide variety of proteins and factors secreted by porcine Sertoli cells benefit the survival and maturation of embryonic DA neurons and suggest that cotransplantation of Sertoli cells and embryonic DA neurons may be useful for a cell transplantation therapy in PD.

The X-gal caution in neural transplantation studies.

Cell transplantation into host brain requires a reliable cell marker to trace lineage and location of grafted cells in host tissue. The lacZ gene encodes the bacterial (E. coli) enzyme beta-galactosidase (beta-gal) and is commonly visualized as a blue intracellular precipitate following its incubation with a substrate, "X gal," in an oxidation reaction. LacZ is the "reporter gene" most commonly employed to follow gene expression in neural tissue or to track the fate of transplanted exogenous cells. If the reaction is not performed carefully-with adequate optimization and individualization of various parameters (e.g.. pH, concentration of reagents, addition of chelators, composition of fixatives) and the establishment of various controls--then misleading nonspecific background X-gal positivity can result, leading to the misidentification of cells. Some of this background results from endogenous nonbacterial beta-gal activity in discrete populations of neurons in the mammalian brain; some results from an excessive oxidation reaction. Surprisingly, few articles have empha sized how to recognize and to eliminate these potential confounding artifacts in order to maximize the utility and credibility of this histochemical technique as a cell marker. We briefly review the phenomenon in general, discuss a specific case that illustrates how an insufficiently scrutinized X-gal positivity can be a pitfall in cell transplantation studies, and then provide recommendations for optimizing the specificity and reliability of this histochemical reaction for discerning E. coli beta-gal activity.

Microcarrier enhanced survival of human and rat fetal ventral mesencephalon cells implanted in the rat striatum.

The transplantation of tissue containing dopamine-producing cells into the mammalian central nervous system is an emerging treatment for Parkinson's disease, despite relatively poor survival of implanted tissue. Recent evidence has suggested that Cytodex microcarriers enhance the survival of dopaminergic rat chromaffin cells transplanted into the rat striatum in the absence of immunosuppression. The current study was undertaken to evaluate the survival of rat and human fetal ventral mesencephalic neurons (VM) implanted alone or after attachment to microcarriers in the striatum of rats without immunosuppression. Rat fetal VM neurons demonstrated enhanced survival in the rat striatum when transplanted on microcarriers, compared to their transplantation alone during the 3-mo period examined in the present study. Transplants of human fetal VM neurons on microcarriers also survived remarkably well in the rat striatum without systemic immunosuppression. In contrast, human fetal VM cells transplanted alone into the rat striatum did not survive without systemic immunosuppression. There was no evidence of TH fiber sprouting in the vicinity of any transplant site. These data indicated that Cytodex microcarriers provide enhanced survival of both rat allograft and human xenograft fetal mesencephalic cells in the rat striatum without the necessity of systemic immunosuppression, perhaps by inducing a unique neuron-glia environment.

Enhancement of horseradish peroxidase histochemistry and/or uptake with radiocontrast media.

The effects of dissolving horseradish peroxidase (HRP) or HRP conjugated to wheatgerm agglutinin (WGA-HRP) in radiocontrast media (MD76) on the intraaxonal transport and enzymatic activity of these tracers were evaluated both in vivo and in vitro. The in vivo studies showed that more reaction product was visible in the L4 dorsal horn following soaking of the sciatic or peroneal nerve, or following cutaneous injection of WGA-HRP, when WGA-HRP was dissolved in MD76, as compared to dissolving WGA-HRP in distilled water. Additionally, there appeared to be an enhancement of anterograde transport and a reduction of retrograde transport of WGA-HRP when this tracer was dissolved in MD76, as compared to dissolving it in distilled water. The in vitro studies indicated that radiocontrast media increased the enzymatic activity of both WGA-HRP and HRP as compared to their enzymatic activity in distilled water when assayed spectrophotometrically. Data are presented that indicate some binding (about 4%) of HRP with MD76. This binding of HRP with organically bound iodine may account for the enhancement of anterograde transport and/or increased enzymatic activity.

The organization of projections to selected points of somatosensory cortex from the cat ventrobasal complex.

The organization of neurons in the cat ventrobasal complex (VB) which project to somatosensory cortex (SI) was investigated by the use of the retrograde transport of the enzyme horseradish peroxidase (HRP). Two histochemical procedures were used to visualize retrogradely transported HRP. Injections of HRP in electrophysiologically characterized points of SI cortex labeled distinctive zones of neurons in VB ipsilateral to the injections. Injections placed in the forelimb or hindlimb cortical areas labeled laminar-like aggregates of neurons have long axis corresponded to the long axis of VB. Injections of the SI trigeminal representation resulted in very compact aggregates of HRP positive neurons which were less clearly laminar. The density of projection from VB to various portions of SI paralleled the general innervation density of the peripheral skin. Injections of the cortical vibrissal, face and forepaw representations labeled a greater number of neurons in VB per unit area of cortex injected than did injections of the hindpaw or trunk representations. For a given somatotopic area, the number of labeled neurons in VB increased linearly as the area of the cortical HRP injection increased. Differences in the sensitivity of each histochemical procedure and the relationship of differing sensitivities to the observed results are also discussed.

The release of pituitary LH and sprouting of LH-releasing hormone (LHRH)-containing neurons after anterior hypothalamic deafferentation.

The chronology of changes in plasma luteinizing hormone (LH) and the distribution of immunoreactive neuronal processes containing LH releasing hormone (LHRH-ir) were studied in the female rat after surgical interruption of anterior neural connections of the mediobasal hypothalamus (MBH). Spontaneous LH surges on the afternoon of proestrus and LH release after estradiol benzoate (EB) followed 48 h later by progesterone (P) administration were studied in ovariectomized (OVX) rats. The maximum increase in plasma concentrations (delta maxLH) after EBP was calculated for each rat at several intervals over 140 days. Control animals given EBP at monthly intervals after OVX had comparably large delta maxLH surges during the first few months of study. However, a gradual decline in control delta maxLH followed becoming significant 3 months after the start of the experiment. In contrast, frontal cuts (FC), which interrupted anterior MBH connections, produced an abrupt decrease in delta maxLH surges after EBP to 11% of preoperative levels. However, during subsequent EBP trials, there was a gradual improvement in LH surges to about 50% of preoperative levels over 100 days. In some cases, individual improvement became equal to preoperative LH surge levels, in others there was no recovery. Examination of LHRH-ir nerve fiber growth responses after FC suggested that sprouting by these peptide-containing neuronal processes may have contributed to the functional improvements observed.