Targeted cell reprogramming produces analgesic chromaffin-like cells from human mesenchymal stem cells.

Transplantation of allogeneic adrenal chromaffin cells demonstrated the promise of favorable outcomes for pain relief in patients. However, there is a very limited availability of suitable human adrenal gland tissues, genetically well-matched donors in particular, to serve as grafts. Xenogeneic materials, such as porcine and bovine adrenal chromaffin cells, present problems; for instance, immune rejection and possible pathogenic contamination are potential issues. To overcome these challenges, we have tested the novel approach of cell reprogramming to reprogram human bone marrow (BM)-derived mesenchymal stem cells (hMSCs) using cellular extracts of porcine chromaffin cells. We produced a new type of cell, chromaffin-like cells, generated from the reprogrammed hMSCs, which displayed a significant increase in expression of human preproenkephalin (hPPE), a precursor for enkephalin opioid peptides, compared to the inherent expression of hPPE in naive hMSCs. The resultant chromaffin-like cells not only expressed the key molecular markers of adrenal chromaffin cells, such as tyrosine hydroxylase (TH) and methionine enkephalin (Met-enkephalin), but also secreted opioid peptide Met-enkephalin in culture. In addition, intrathecal injection of chromaffin-like cells in rats produced significant analgesic effects without using immunosuppressants. These results suggest that analgesic chromaffin-like cells can be produced from an individual's own tissue-derived stem cells by targeted cell reprogramming and also that these chromaffin-like cells may serve as potential autografts for chronic pain management.

GABAA receptors and benzodiazepines: a role for dendritic resident subunit mRNAs.

This review is designed to describe the evolution of the seminal observation made simultaneously in 1975 by Dr. W. Haefely's laboratory (Hoffman La Roche, Basel, Switzerland) and in the Laboratory of Preclinical Pharmacology (NIH, St. Elizabeths Hospital, Washington DC), that benzodiazepine action was mediated by a modulation of GABA action at GABA(A) receptors. In fact, our suggestion was that the benzodiazepine receptor was "a receptor on a receptor" and that this receptor was GABA(A). Needless to say, this suggestion created opposition, but we did not abandon the original idea, in fact, as shown in this review, there is now universal agreement with our hypothesis on the mode of action of benzodiazepines. Hence, this review deals with the allosteric modulation of GABA(A) receptors by benzodiazepines, the role of GABA(A) receptors and benzodiazepine structure diversities in this modulation, and describes the results of our attempts to establish a benzodiazepine (imidazenil) devoid of tolerance, withdrawal symptoms, and changes in the expression of GABA(A) receptor subunits during tolerance. It also deals with the idea that the synthesis of GABA(A) receptor subunits triggered by tolerance resides in dendrites and spines where mRNAs and the apparatus for this translation is located. New analytic procedures may foster progress in the understanding of tolerance to and withdrawal from benzodiazepines.

New structure involved in transient membrane fusion and exocytosis.

Our studies using atomic force microscopy (AFM) reveal a new group of plasma membrane structures involved in exocytosis in live pancreatic acinar cells. These studies demonstrate that "pits" and "depressions" are sites at the apical plasma membrane in live cells, where membrane-bound secretory vesicles dock and transiently fuse to release vesicular contents.

Reelin function in neural stem cell biology.

In the adult brain, neural stem cells (NSC) must migrate to express their neuroplastic potential. The addition of recombinant reelin to human NSC (HNSC) cultures facilitates neuronal retraction in the neurospheroid. Because we detected reelin, alpha3-integrin receptor subunits, and disabled-1 immunoreactivity in HNSC cultures, it is possible that integrin-mediated reelin signal transduction is operative in these cultures. To investigate whether reelin is important in the regulation of NSC migration, we injected HNSCs into the lateral ventricle of null reeler and wild-type mice. Four weeks after transplantation, we detected symmetrical migration and extensive neuronal and glial differentiation of transplanted HNSCs in wild-type, but not in reeler mice. In reeler mice, most of the injected HNSCs failed to migrate or to display the typical differentiation pattern. However, a subpopulation of transplanted HNSCs expressing reelin did show a pattern of chain migration in the reeler mouse cortex. We also analyzed the endogenous NSC population in the reeler mouse using bromodeoxyuridine injections. In reeler mice, the endogenous NSC population in the hippocampus and olfactory bulb was significantly reduced compared with wild-type mice; in contrast, endogenous NSCs expressed in the subventricular zonewere preserved. Hence, it seems likely that the lack of endogenous reelin may have disrupted the migration of the NSCs that had proliferated in the SVZ. We suggest that a possible inhibition of NSC migration in psychiatric patients with a reelin deficit may be a potential problem in successful NSC transplantation in these patients.

Porocytosis: a new approach to synaptic function.

We propose a new approach to address the question of how a single quantum of neurotransmitter is secreted from a presynaptic terminal whose clustered secretory vesicles are locally bathed in high levels of calcium ions [Proceedings of the Symposium on Bioelectrogenesis (1961) 297-309; The Physiology of Synapses (1964) Chapters 1, 4, 5, 6; How the Self Controls its Brain (1994) Chapters 1, 4, 5, 6; Science 256 (1992) 677-679]. This hypothesis, which we term 'porocytosis', posits that the post-synaptic quantal response results from transmitter secreted through an array of docked vesicle/secretory pore complexes. The transient increase in calcium ions, which results from the voltage activated calcium channels, stimulates the array of secretory pores to simultaneously flicker open to pulse transmitter. Porocytosis is consistent with the quantal nature of presynaptic secretion and transmission, and with available biochemical, morphological and physiological evidence. It explains the frequency dependency of quantal size as a function of the secretion process. It permits a signature amount of transmitter release for different frequencies allowing a given synapse to be employed in different behavioral responses. The porocytosis hypothesis permits fidelity of secretion and the seemingly apposed characteristic of synaptic plasticity. The dynamics inherent in an array insure a constant quantal size as a function of the number of units within the array. In this hypothesis, plasticity is a consequence of concurrent pre- and post-synaptic changes due to a change in array size. Changes in the number of docked vesicle-secretory pore complexes composing the array can explain facilitation, depletion, graded excitation-secretion and long term plasticity.

Dendritic spine hypoplasticity and downregulation of reelin and GABAergic tone in schizophrenia vulnerability.

In this review, we will first present a brief overview of the current understanding of: (a) the biology of reelin; (b) the putative reelin signaling pathways via integrin receptor stimulation; (c) the cytosolic adapter protein DAB1, which appears to be operative in the transduction of reelin's pleiotropic actions in embryonic, adolescent, and adult brain; (d) the regulation of GABAergic function, including some aspects of GABAergic system development; and (e) dendritic spine function and its role in the regulation of synaptic plasticity. We argue that a downregulation of reelin expression occurring in prefrontal cortex and in every brain structure of schizophrenia patients so far studied may be associated with a decrease in dendritic spine expression that in turn may provide an important reduction of cortical function as documented by the downregulation of glutamic acid decarboxylase67 (GAD67) expression, which might be secondary to a reduction of GABAergic axon terminals. This hypothesis is supported by a genetic mouse model of reelin haploinsufficiency that replicates the above-described dendritic and presynaptic GABAergic defects documented in schizophrenia brains.

Reelin in the extracellular matrix and dendritic spines of the cortex and hippocampus: a comparison between wild type and heterozygous reeler mice by immunoelectron microscopy.

Reelin is a glycoprotein ( approximately 400 kDa) secreted by GABAergic neurons into the extracellular matrix of the neocortex and hippocampus as well as other areas of adult rodent and nonhuman primate brains. Recent findings indicate that the heterozygote reeler mouse (haploinsufficient for the reeler gene) shares several neurochemical and behavioral abnormalities with schizophrenia and bipolar disorder with mania. These include (1) a downregulation of both reelin mRNA and the translated proteins, (2) a decrease in the number of dendritic spines in cortical and hippocampal neurons, (3) a concomitant increase in the packing density of cortical pyramidal neurons, and (4) an age-dependent decrease in prepulse inhibition of startle. Interestingly, the heterozygous reeler mouse does not exhibit the unstable gait or the neuroanatomy characteristic of the null mutant reeler mouse. Immunocytochemical studies of the expression of reelin in mice have been primarily limited to light microscopy. In this study we present new immunoelectron microscopy data that delineates the subcellular localization of reelin in the cortex and hippocampus of the wild-type mouse, and compares these results to reelin expression in the heterozygous reeler mouse. In discontinuous areas of cortical layers I and II and the inner blade area of the dentate gyrus of the wild type mouse, extracellular reelin is associated with dendrites and dendritic spine postsynaptic specializations. Similar associations have been detected in the CA1 stratum oriens and other areas of the hippocampus. In the hippocampus, reelin expression is more expansive and more widespread than in cortical layers I and II. In contrast, extracellular reelin immunoreactivity is greatly diminished in all areas examined in the heterozygous reeler mouse. However, some cell bodies of GABAergic neurons in the cortex and hippocampus demonstrate an increased accumulation of reelin in the Golgi and endoplasmic reticulum. We suggest that in the heterozygous reeler mouse a downregulation of reelin biosynthesis results in a decreased rate of secretion into the extracellular space. This inhibits dendritic spine maturation and plasticity and leads to dissociation of dendritic postsynaptic density integrity and atrophy of spines. We speculate that the haploinsufficient reeler mouse may provide a model for future studies of the role of reelin, as it may be related to psychosis vulnerability.

Colocalization of integrin receptors and reelin in dendritic spine postsynaptic densities of adult nonhuman primate cortex.

The expression of telencephalic reelin (Reln) and glutamic acid decarboxylase mRNAs and their respective cognate proteins is down-regulated in postmortem brains of schizophrenia and bipolar disorder patients. To interpret the pathophysiological significance of this finding, immunoelectron microscopic experiments are required, but these cannot be carried out in postmortem human brains. As an alternative, we carried out such experiments in the cortex of rats and nonhuman primates. We found that Reln is expressed predominantly in layer I of both cortices and is localized to bitufted (double-bouquet), horizontal, and multipolar gamma-aminobutyric acid-ergic interneurons, which secrete Reln into extracellular matrix. Reln secretion is mediated by a constitutive mechanism that depends on the expression of a specific signal peptide present in the Reln carboxy-terminal domain. Extracellular matrix Reln is found to aggregate in proximity of postsynaptic densities expressed in apical dendrite spines, which include also the alpha(3) subunit of integrin receptors. Most pyramidal neurons of various cortical layers express the mouse-disabled 1 (Dab1) protein, which, after phosphorylation by a soluble tyrosine kinase, functions as an adapter protein, probably mediating a modulation of cytoskeleton protein expression. We hypothesize that the decrease of neuropil and dendritic spine density reported to exist in the neocortex of psychiatric patients may be related to a down-regulation of Reln-integrin interactions and the consequent decrease of cytoskeleton protein turnover.

Expression of reelin in adult mammalian blood, liver, pituitary pars intermedia, and adrenal chromaffin cells.

Reelin regulates telencephalic and cerebellar lamination during mammalian development and is expressed in several structures of the adult brain; however, only traces of reelin were believed to be in peripheral tissues. Because reelin structurally resembles extracellular matrix proteins, and because many of these proteins are expressed in blood, we hypothesized that reelin also might be detectable in the circulation. Reelin (420 kDa) and two reelin-like immunoreactive bands (310 and 160 kDa) are expressed in serum and platelet-poor plasma of rats, mice, and humans, but these three bands were not detectable in serum of homozygous reeler (rl/rl) mice. Reelin plasma levels in heterozygous (rl/+) mice were half of those in wild-type littermates. Western blotting and immunocytochemistry using antireelin mAbs indicated that reelin-like immunoreactivity was expressed in a subset of chromaffin cells within the rat adrenal medulla and in a subset of cells coexpressing alpha-melanocyte-stimulating hormone within the pituitary pars intermedia. However, surgical removal of adrenal or pituitary failed to decrease the amount of reelin (420-kDa band) expressed in serum. Adult liver expressed one-third of the reelin mRNA concentration expressed in adult mouse cerebral cortex. Full-length reelin protein was detectable in liver extracts in situ; acutely isolated liver cells also secreted full-length reelin in vitro. Liver appears to be a prime candidate to produce and maintain the circulating reelin pool. It now becomes relevant to ask whether circulating reelin has a physiologic role on one or more peripheral target tissues.

Comparison of tyrosine hydroxylase and preproenkephalin expression in rat adrenal medullary explants in vitro and transplanted into subarachnoid space.

When adrenal medullary cells are cultured in vitro, tyrosine hydroxylase (TH) mRNA, preproenkephalin (PPEnk) mRNA, and methionine enkephalin (Mek) immunoreactivity was markedly increased compared with intact adrenal medullary cells in situ, suggesting an increased biosynthesis of catecholamines and enkephalin-containing peptides. In transplanted adrenal medullary cells in vivo, TH mRNA and TH immunoreactivity are still apparent for at least 1 year after transplantation, indicating continued capacity for catecholamine biosynthesis. PPEnk mRNA levels in surviving adrenal medullary grafted cells increased, particularly in the first week after transplantation, and remained above levels found in the intact adrenal gland for at least 1 year after transplantation. These results support other studies in our laboratory, suggesting that adrenal medullary transplants reduce pain by synthesis and secretion of both catecholamines and enkephalin-containing peptides. The differences in expression of TH mRNA and PPEnk mRNA in the adrenal medulla in situ, in explants in culture and in transplants in the spinal subarachnoid space, indicate that the mechanisms regulating the expression of neurohumoral factors depend upon environmental factors extrinsic to the medullary cells themselves.

Purification of adrenal chromaffin cells increases antinociceptive efficacy of xenotransplants without immunosuppression.

We have found that immunosuppression is necessary for the survival of xenogeneic adrenal medullary transplants. Because chromaffin cells are essentially nonimmunogenic, it is likely that the highly immunogenic "passenger" cells in the transplant preparation bring about rejection. This article describes a procedure that produces an essentially pure preparation of chromaffin cells for transplantation. Bovine adrenal medullary cells were isolated and differentially plated, resulting in a semipurified preparation of chromaffin cells. Ferromagnetic beads were added to the cell suspension, some of which were phagocytized by endothelial cells, which allowed their removal by exposure to a magnet. The remaining cells were then exposed to ferromagnetic beads coated with isolectin B4 from Griffonia simplicifolia and once again to a magnetic field. The "semipurified" preparation contained approximately 90% chromaffin cells, whereas the "highly purified" preparation was > 99.5% chromaffin cells as determined immunohistochemically. The immunogenicity of the two cell preparations was assessed in vitro by determining their capacity to evoke lymphocyte proliferation. Rat spleen lymphocytes were mixed with either a highly purified or semipurified population of bovine chromaffin cells. The results of this assay demonstrated that the highly purified preparation was a much weaker stimulant of lymphocyte proliferation than was the semipurified preparation and may demonstrate better graft survival in vivo. Transplantation via intrathecal catheter of either 80,000 or 250,000 cells from the highly or partially purified preparations onto the lumbar spinal cord of nonimmunosuppressed and non-nicotine-stimulated rats produced a cell number-dependent antinociception for both A(delta) and C fiber-mediated thermonociception at 6 days after transplantation. After 6 days and up to 28 days, only the "highly purified" preparation showed antinociception. These results suggest that nearly complete purification of bovine chromaffin cells minimizes immunorejection of xenogeneic transplants of these cells.

Fine structure of host-graft relationships between transplanted chromaffin cells and CNS.

Our laboratory studies have shown that transplantation of adrenal medullary tissue or isolated chromaffin cells into central nervous system (CNS) pain modulatory regions (i.e., periaqueductal gray and subarachnoid lumbar spinal cord) can reduce pain sensitivity of rats in both acute and chronic pain. The analgesia produced by these transplants is thought to result from release of both opiate peptides and catecholamines. Morphologically, these animal studies also suggest that there is no development of tolerance over long periods of time, and the transplanted chromaffin cells appear to be robust and well integrated with the host tissue. In our initial clinical studies, where allografts of adrenal medullary tissue were transplanted intrathecally to relieve intractable cancer pain, patients obtained significant and long-lasting pain relief. Increased cerebrospinal fluid (CSF) levels of metenkephalin were correlated with the decreased pain scores. Histology of autopsy tissue obtained from two patients with 1 year transplants revealed viable transplanted chromaffin cells. Because of the limited availability of human adrenal glands, sources of xenogeneic chromaffin cells will need to be identified if effective transplantation therapy for chronic pain is to be developed further.

A decrease of reelin expression as a putative vulnerability factor in schizophrenia.

Postmortem prefrontal cortices (PFC) (Brodmann's areas 10 and 46), temporal cortices (Brodmann's area 22), hippocampi, caudate nuclei, and cerebella of schizophrenia patients and their matched nonpsychiatric subjects were compared for reelin (RELN) mRNA and reelin (RELN) protein content. In all of the brain areas studied, RELN and its mRNA were significantly reduced (approximately 50%) in patients with schizophrenia; this decrease was similar in patients affected by undifferentiated or paranoid schizophrenia. To exclude possible artifacts caused by postmortem mRNA degradation, we measured the mRNAs in the same PFC extracts from gamma-aminobutyric acid (GABA)A receptors alpha1 and alpha5 and nicotinic acetylcholine receptor alpha7 subunits. Whereas the expression of the alpha7 nicotinic acetylcholine receptor subunit was normal, that of the alpha1 and alpha5 receptor subunits of GABAA was increased when schizophrenia was present. RELN mRNA was preferentially expressed in GABAergic interneurons of PFC, temporal cortex, hippocampus, and glutamatergic granule cells of cerebellum. A protein putatively functioning as an intracellular target for the signal-transduction cascade triggered by RELN protein released into the extracellular matrix is termed mouse disabled-1 (DAB1) and is expressed at comparable levels in the neuroplasm of the PFC and hippocampal pyramidal neurons, cerebellar Purkinje neurons of schizophrenia patients, and nonpsychiatric subjects; these three types of neurons do not express RELN protein. In the same samples of temporal cortex, we found a decrease in RELN protein of approximately 50% but no changes in DAB1 protein expression. We also observed a large (up to 70%) decrease of GAD67 but only a small decrease of GAD65 protein content. These findings are interpreted within a neurodevelopmental/vulnerability "two-hit" model for the etiology of schizophrenia.

Neural tissue engineering: adrenal chromaffin cell attachment and viability on chitosan scaffolds.

This study introduces chitosan-based matrices as cell substrates for bovine chromaffin cell attachment in transplantation procedures. Chitosan ([1-->4] linked 2-amino-2-deoxy-beta-D-glucopyranose), having structural similarity to glycosaminoglycans, was modified using several proteins (collagen, albumin and gelatin) to increase surface area and improve biocompatibility. In vitro, collagen-blended chitosan (CC) matrices were found to attach more readily to chromaffin cells than to gelatin- or albumin-blended matrices. Morphological evidence showed that the chromaffin cells attached to CC substrates integrated well with the hydrogel matrix and survived for at least two weeks, under in vivo culture conditions. The chromaffin cells within chitosan scaffolds also survived for at least two weeks in vitro and after subarachnoid grafting to rats.

One-year chromaffin cell allograft survival in cancer patients with chronic pain: morphological and functional evidence.

The control of chronic pain through transplantation of chromaffin cells has been reported over the past few years. Analgesic effects are principally due to the production of opioid peptides and catecholamines by chromaffin cells. Clinical trials have been reported with allografts consisting of whole-tissue fragments implanted into the subarachnoid space of the lumbar spinal cord (14,19,36). In the present study, allogeneic grafts were successfully used to control chronic pain in two patients over a period of 1 yr based on patient reported pain scores, morphine intake, and CSF levels of Met-enkephalin. Macroscopic examination at autopsy located the transplanted tissue fragments in the form of multilobulated nodules at the level of the spinal axis and cauda equina. Immunocytochemical microscopy showed neuroendocrine cells are positive for chromagranin A (CGA), and enzymes tyrosine hydroxylase (TH) and dopamine-beta-hydroxylase (DbetaH). The results suggest that there is a relationship between analgesic effect, Met-enkephalin levels in CSF, and the presence of chromaffin cells surviving in spinal subarachnoid space.

The influence of xenotransplant immunogenicity and immunosuppression on host MHC expression in the rat CNS.

During the early stages following neural transplantation, host immune responses are initiated that are not normally found in the CNS including the induction of major histocompatibility antigens (MHC I and II). Previous laboratory findings have demonstrated prolonged survival of bovine chromaffin cells (BCC) in the rat CNS following transient immunosuppression with cyclosporin A (CSA) providing chromaffin cells are isolated from highly immunogenic passenger cells. To assess the influence of passenger and chromaffin cells on host MHC I and II expression, either BCC, nonchromaffin cell adrenal constituents (NCC), or adrenal medullary endothelial cells (EC) were implanted into the host. At 2 weeks postimplantation, robust BCC survival was obtained in CSA-treated animals. This correlated with low expression of MHC I at the host-graft border and the virtual absence of MHC II. Good BCC survival with reduced MHC I expression only was seen at 6 weeks postimplantation in animals transiently immunosuppressed (4 weeks). In contrast, poor survival was seen in the EC group (even with CSA treatment). In addition, marked MHC I and II expression was found in and around these grafts at 2 weeks, and was particularly intense in EC implanted animals. The results of this study suggest that nonchromaffin passenger cells in BCC preparations, most notably endothelial cells, can induce strong immune responses even in the presence of immunosuppression. Based on MHC staining, removal of these passenger cells can reduce host responses and improve long term survival of xenogeneic chromaffin cells in the CNS.

A subpopulation of reactive astrocytes at the immediate site of cerebral cortical injury.

We have identified an early-appearing intermediate filament-associated protein (IFAP-70/280 kDa) in radial glia and their immediate derivatives. This IFAP is absent in the adult CNS. In this study, we examined the reexpression of this early glial differentiation trait in rat reactive astrocytes induced by stab injury of the cerebrum. Double-label immunofluorescence microscopy demonstrated that by 36 h postlesion, IFAP-70/280 kDa was present in a few GFAP-positive astrocytes in the area adjacent to the wound. As the gliotic reaction progressed, the number of IFAP-positive reactive astrocytes increased and by 5-6 days postlesion, IFAP-70/280 kDa was present in most of the hypertrophied astrocytes in tissue immediately adjacent to the wound. By 8 days postlesion, while the number of IFAP-negative reactive astrocytes away from the wound diminished, the IFAP-containing reactive astrocytes close to the wound persisted. Concurrently, they began to change from a stellate form to an elongated shape, with their longitudinal axes radiating from the wound. The immunoreactivity of this IFAP started to diminish at 20 days postlesion, and by 30 days postlesion, it was not observed in the remaining gliotic cells. These results demonstrate that reactive astrocytes induced by stab-wound injury can be divided into two subtypes: persistent IFAP-70/280 kDa-containing cells which are close to the wound in the area of the glial scar and transient IFAP-70/280 kDa-negative cells which are farther from the wound. The reappearance of IFAP-70/280 kDa also suggests that some reactive astrocytes have the capacity to recapitulate early developmental stages.

Relief of intractable cancer pain by human chromaffin cell transplants: experience at two medical centers.

In addition to its possible role as a replacement source in CNS degenerative diseases, neural transplantation may be used to augment the normal production of neuroactive substances. Our laboratory at the University of Illinois at Chicago has shown, in both acute and chronic pain models, that transplantation of adrenal medullary tissue or isolated chromaffin cells into CNS pain modulatory regions can reduce pain sensitivity in rodents. Chromaffin cells were chosen as the donor source since they produce high levels of both opioid peptides and catecholamines, substances which reduce pain sensitivity when injected locally into the spinal subarachnoid space. The analgesia produced by these transplants probably results from the release of both opioid peptides and catecholamines since it can be blocked or attenuated by both opiate and adrenergic antagonists. Studies indicate that even over long periods there is no apparent development of tolerance. Promising results have been obtained in preliminary clinical studies using allografts of adrenal medulla to relieve cancer pain. This clinical review encompasses results at two Medical Centers-University of Illinois at Chicago and University Paul Sabatier, Toulouse, France-in assessing efficacy of subarachnoid adrenal medullary transplantation for alleviating cancer pain. Our clinical and autopsy data strongly support our previous laboratory studies, i.e., that chromaffin cell transplants into the subarachnoid space represent a promising new approach to the alleviation of chronic pain. It is suggested that further clinical studies are now warranted.

Loss of GABA-immunoreactivity in the spinal dorsal horn of rats with peripheral nerve injury and promotion of recovery by adrenal medullary grafts.

Abnormal pain-related behaviour that accompanies peripheral nerve injury may be the result of altered spinal neuronal function. The long-term loss of inhibitory function by GABA neurons in particular may be a mechanism by which abnormal neural hyperactivity occurs, leading to exaggerated sensory processing following nerve injury. In order to assess this, changes in spinal GABA immunoreactivity at several time points following constriction nerve injury were quantified in parallel with behavioural assessments of abnormal sensory responses to noxious and innocuous stimuli. In addition, the effects of spinal adrenal medullary transplants were determined since previous findings have demonstrated alleviation of behavioural pain symptoms by such transplants. In response to unilateral sciatic nerve injury, GABAergic profiles normally found in lumbar dorsal horn laminae I-III significantly decreased. The decrease was apparent three days following ligation, particularly on the side ipsilateral to the nerve injury. By two weeks, no GABAergic profiles could be seen, with the deficit appearing in the spinal dorsal horn both ipsilateral and contralateral to the unilateral peripheral nerve injury. Marked decreases in GABA-immunoreactive profiles persisted for at least up to five weeks post-injury, with partial restoration occurring by seven weeks. However, even at seven weeks, losses in GABA-immunoreactive profiles persisted in the dorsal horn ipsilateral to peripheral nerve injury. These findings were comparable in animals receiving control striated muscle transplants. In contrast, adrenal medullary transplants markedly reduced the loss in GABA-immunoreactive profiles at all time-points examined. In addition, GABA-immunoreactive profile levels were normalized near that of intact animals by five to seven weeks following nerve injury in animals with adrenal medullary transplants. Parallel improvements in sensory responses to innocuous and noxious stimuli were also observed in these animals. The results of this study indicate that peripheral nerve injury can result in severe losses in spinal inhibitory mechanisms, possibly leading to exaggerated sensory processes in persistent pain states. In addition, adrenal medullary transplants may provide a neuroprotective function in promoting recovery and improving long-term survival of GABAergic neurons in the spinal dorsal horn which have been damaged by excitotoxic injury.