Higher throughput workflow with sensitive, reliable and automatic quantification of myelination in vitro suitable for drug screening.

Multiple sclerosis (MS) is the most common demyelinating autoimmune disease of the central nervous system (CNS). Immune-mediated myelin and axonal damage that is accompanied by chronic axonal loss causing destruction of the myelin sheaths are hallmarks of MS. While great strides have been made in understanding the molecular underpinnings of re-/myelination, currently no remyelination therapy is available for MS. As myelination is a complex process that is not fully understood, we sought to develop a systematic, reliable, automated and quantitative higher throughput screening method. We aimed to quantitate myelin sheaths in vitro with high sensitivity at the single cell level suitable for testing small compound libraries. To this end, we miniaturised in vitro retinal ganglion cell-oligodendrocyte precursor cell (RGC-OPC) co-cultures into a multi-well plate format. This allowed us to maintain the reciprocal interaction of live axons and oligodendrocytes (OLs) to ensure compact myelin formation. To quantify our co-cultures, we developed a novel computer vision algorithm to precisely measure myelination. We demonstrated efficacy of our system with known pro-differentiating compounds BQ3020 and XAV939 which exhibited robust, efficient, and dose dependent effects on myelination. Through this combination of experimental and technical advances, we have developed a method allowing systematic and reliable testing of remyelinating compound efficacy.

14-3-3 signal adaptor and scaffold proteins mediate GPCR trafficking.

Receptor trafficking is pivotal for the temporal and spatial control of GPCR signaling and is regulated by multiple cellular proteins. We provide evidence that GPCRs interact with 14-3-3 signal adaptor/scaffold proteins and that this interaction regulates receptor trafficking in two ways. We found GPCR/14-3-3 interaction signals can be agonist-induced or agonist-inhibited. Some GPCRs associate with 14-3-3 proteins at the cell membrane and agonist treatments result in disrupted GPCR/14-3-3 interaction signals. The diminished GPCR/14-3-3 interaction signals are temporally correlated with increased GPCR/ß-arrestin interaction signals in response to agonist treatment. Other GPCRs showed agonist-induced GPCR/14-3-3 interaction signal increases that occur later than agonist-induced GPCR/ß-arrestin interaction signals, indicating that GPCR/14-3-3 interaction occurred after receptor endocytosis. These two types of GPCR/14-3-3 interaction patterns correlate with different receptor trafficking patterns. In addition, the bioinformatic analysis predicts that approximately 90% of GPCRs contain at least one putative 14-3-3 binding motif, suggesting GPCR/14-3-3 association could be a general phenomenon. Based on these results and collective evidence, we propose a working model whereby 14-3-3 serves as a sorting factor to regulate receptor trafficking.

Detection of Concordance between Transcriptional Levels of GPCRs and Receptor-Activity-Modifying Proteins.

A recent phylogenetic analysis showed global co-evolution of G protein-coupled receptors (GPCRs) and receptor-activity-modifying proteins (RAMPs) suggesting global interactions between these two protein families. Experimental validation of these findings is challenging because in humans whereas there are only three genes encoding RAMPs, there are about 800 genes encoding GPCRs. Here, we report an experimental approach to evaluate GPCR-RAMP interactions. As a proof-of-concept experiment, we over-expressed RAMP2 in HEK293T cells and evaluated the effect on the transcriptional levels of 14 representative GPCRs that were selected based on the earlier phylogenetic analysis. We utilized a multiplexed error-correcting fluorescence in situ hybridization (MERFISH) method to detect message levels for individual GPCRs in single cells. The MERFISH results showed changes in GPCR message levels with RAMP2 over-expression in a concordant pattern that was predicted by the earlier phylogenetic analysis. These results provide additional evidence that GPCR-RAMP interactions are more widespread than previously appreciated and that these interactions have functional consequences.

Brain gene expression signature on primate genomic sequence evolution.

Considering the overwhelming changes that occurred during primate evolution in brain structure and function, one might expect corresponding changes at the molecular level. Surprisingly, a relatively constrained gene expression pattern is observed in brain compared with other tissues among primates, an observation that calls for reassessment of RNA expression influence on primate genome evolution. We built phylogenetic trees based on genomic sequences of functional genomic regions and tissue-specific RNA expression in eight tissue types for six primate species. Comparisons of the phylogenetic trees from brain tissues revealed that DNA- and RNA-based trees were significantly similar. The similarity was specific for promoter regions and cerebellum and frontal cortex expression, suggesting a major impact of gene regulation in the brain on genome shaping along the primate branch.

GPCRs globally coevolved with receptor activity-modifying proteins, RAMPs.

Receptor activity-modifying proteins (RAMPs) are widely expressed in human tissues and, in some cases, have been shown to affect surface expression or ligand specificity of G-protein-coupled receptors (GPCRs). However, whether RAMP-GPCR interactions are widespread, and the nature of their functional consequences, remains largely unknown. In humans, there are three RAMPs and over 800 expressed GPCRs, making direct experimental approaches challenging. We analyzed relevant genomic data from all currently available sequenced organisms. We discovered that RAMPs and GPCRs tend to have orthologs in the same species and have correlated phylogenetic trees to the same extent, or higher than other interacting protein pairs that play key roles in cellular signaling. In addition, the resulting RAMP-GPCR interaction map suggests that RAMP1 and RAMP3 interact with the same set of GPCRs, which implies functional redundancy. We next analyzed human transcriptomes and found expression correlation for GPCRs and RAMPs. Our results suggest global coevolution of GPCRs and RAMPS and support the hypothesis that GPCRs interact globally with RAMPs in cellular signaling pathways.

Alzheimer's brains show inter-related changes in RNA and lipid metabolism.

Alzheimer's disease (AD) involves changes in both lipid and RNA metabolism, but it remained unknown if these differences associate with AD's cognition and/or post-mortem neuropathology indices. Here, we report RNA-sequencing evidence of inter-related associations between lipid processing, cognition level, and AD neuropathology. In two unrelated cohorts, we identified pathway-enriched facilitation of lipid processing and alternative splicing genes, including the neuronal-enriched NOVA1 and hnRNPA1. Specifically, this association emerged in temporal lobe tissue samples from donors where postmortem evidence demonstrated AD neuropathology, but who presented normal cognition proximate to death. The observed changes further associated with modified ATP synthesis and mitochondrial transcripts, indicating metabolic relevance; accordingly, mass-spectrometry-derived lipidomic profiles distinguished between individuals with and without cognitive impairment prior to death. In spite of the limited group sizes, tissues from persons with both cognitive impairment and AD pathology showed elevation in several drug-targeted genes of other brain, vascular and autoimmune disorders, accompanied by pathology-related increases in distinct lipid processing transcripts, and in the RNA metabolism genes hnRNPH2, TARDBP, CLP1 and EWSR1. To further detect 3'-polyadenylation variants, we employed multiple cDNA primer pairs. This identified variants that showed limited differences in scope and length between the tested cohorts, yet enabled superior clustering of demented and non-demented AD brains versus controls compared to total mRNA expression values. Our findings indicate inter-related cognition-associated differences in AD's lipid processing, alternative splicing and 3'-polyadenylation, calling for pursuing the underlying psychological and therapeutics implications.

Length-dependent gene misexpression is associated with Alzheimer's disease progression.

Recent reports show transcription preference for long genes in neuronal tissues compared with non-neuronal tissues, and a gene-length dependent change in expression in the neurodevelopmental disease Rett syndrome (RTT). Whether the gene-length dependent changes in expression seen in RTT might also be seen in neurodegenerative diseases is not yet known. However, a reasonable hypothesis is that similar effects might be seen in neurodegenerative diseases as well as in RTT since a common general feature of both illnesses involves progressive dysfunction of synapses. Here, we demonstrate a clear length-dependent gene misexpression in the most prevalent neurodegenerative disease, Alzheimer's disease. We show that the effect is associated with disease progression and can be attributed specifically to neurons. In particular, we observed gene length-dependent down regulation on the level of the whole tissue and gene length-dependent up regulation on the level of single cells. Our analysis shows that a gene-length effect on expression can be found in degenerative neurological illnesses, such as Alzheimer's disease. Additional investigation to elucidate the precise mechanism underlying gene-length dependent changes in expression is warranted.

Nucleobindin 1 binds to multiple types of pre-fibrillar amyloid and inhibits fibrillization.

During amyloid fibril formation, amyloidogenic polypeptides misfold and self assemble into soluble pre-fibrillar aggregates, i.e., protofibrils, which elongate and mature into insoluble fibrillar aggregates. An emerging class of chaperones, chaperone-like amyloid binding proteins (CLABPs), has been shown to interfere with aggregation of particular misfolded amyloid peptides or proteins. We have discovered that the calcium-binding protein nuclebindin-1 (NUCB1) is a novel CLABP. We show that NUCB1 inhibits aggregation of islet-amyloid polypeptide associated with type 2 diabetes mellitus, a-synuclein associated with Parkinson's disease, transthyretin V30M mutant associated with familial amyloid polyneuropathy, and Aß42 associated with Alzheimer's disease by stabilizing their respective protofibril intermediates. Kinetic studies employing the modeling software AmyloFit show that NUCB1 affects both primary nucleation and secondary nucleation. We hypothesize that NUCB1 binds to the common cross-ß-sheet structure of protofibril aggregates to "cap" and stabilize soluble macromolecular complexes. Transmission electron microscopy and atomic force microscopy were employed to characterize the size, shape and volume distribution of multiple sources of NUCB1-capped protofibrils. Interestingly, NUCB1 prevents Aß42 protofibril toxicity in a cellular assay. NUCB1-stabilized amyloid protofibrils could be used as immunogens to prepare conformation-specific antibodies and as novel tools to develop screens for anti-protofibril diagnostics and therapeutics.

Cholinergic Surveillance over Hippocampal RNA Metabolism and Alzheimer's-Like Pathology.

The relationship between long-term cholinergic dysfunction and risk of developing dementia is poorly understood. Here we used mice with deletion of the vesicular acetylcholine transporter (VAChT) in the forebrain to model cholinergic abnormalities observed in dementia. Whole-genome RNA sequencing of hippocampal samples revealed that cholinergic failure causes changes in RNA metabolism. Remarkably, key transcripts related to Alzheimer's disease are affected. BACE1, for instance, shows abnormal splicing caused by decreased expression of the splicing regulator hnRNPA2/B1. Resulting BACE1 overexpression leads to increased APP processing and accumulation of soluble Aß1-42. This is accompanied by age-related increases in GSK3 activation, tau hyperphosphorylation, caspase-3 activation, decreased synaptic markers, increased neuronal death, and deteriorating cognition. Pharmacological inhibition of GSK3 hyperactivation reversed deficits in synaptic markers and tau hyperphosphorylation induced by cholinergic dysfunction, indicating a key role for GSK3 in some of these pathological changes. Interestingly, in human brains there was a high correlation between decreased levels of VAChT and hnRNPA2/B1 levels with increased tau hyperphosphorylation. These results suggest that changes in RNA processing caused by cholinergic loss can facilitate Alzheimer's-like pathology in mice, providing a mechanism by which decreased cholinergic tone may increase risk of dementia.

Dynamics of social network structure for Alzheimer and Lymphoma scientific communities.

It is generally assumed that sociology affects scientific progress but specific examples of this assumption are hard to find. We examined this hypothesis by comparing the social network structure and its dynamics over the last 16 years, for two common human diseases; Alzheimer's disease, for which there has been very little therapeutic progress, and Lymphoma, were there has been significant therapeutic progress. We found that the Alzheimer's research community is more interlinked ('dense') and more 'cliquish' than that of Lymphoma and suggest that this could affect its scientific progress.

Global coevolution of human microRNAs and their target genes.

MicroRNAs (miRNAs) have presumably contributed to the emergence of the novel expression patterns, higher brain functions, and skills underlying human evolution. However, it is incompletely understood how new miRNAs have evolved in the human lineage because their initial emergence predictably entailed deleterious consequences due to their powerful multitarget effects. Here, we report genetic variation and conservation parameters for miRNAs and their predicted targets in the genomes of 1,092 humans and 58 additional organisms. We show that miRNAs were evolutionarily more conserved than their predicted binding sites, which were inversely subject to the accumulation of single-nucleotide variations over short evolutionary timescales. Moreover, the predictably "younger" human-specific miRNAs presented lower genetic variation than other miRNAs; their targets displayed higher genetic variation compared with other miRNA targets in diverse human populations; and neuronal miRNAs showed yet lower levels of genetic variation and were found to target more protein-coding genes than nonneuronal miRNAs. Furthermore, enrichment analysis indicated that targets of human-specific miRNAs primarily perform neuronal functions. Specifically, the genomic regions harboring the vertebrate-conserved neuronal miRNA-132 presented considerably higher conservation scores than those of its target genes throughout evolution, whereas both the recently evolved human miRNA-941 and its acquired targets showed relatively low conservation. Our findings demonstrate inversely correlated genetic variation around miRNAs and their targets, consistent with theories of coevolution of these elements and the predicted role attributed to miRNAs in recent human evolution.

Forebrain deletion of the vesicular acetylcholine transporter results in deficits in executive function, metabolic, and RNA splicing abnormalities in the prefrontal cortex.

One of the key brain regions in cognitive processing and executive function is the prefrontal cortex (PFC), which receives cholinergic input from basal forebrain cholinergic neurons. We evaluated the contribution of synaptically released acetylcholine (ACh) to executive function by genetically targeting the vesicular acetylcholine transporter (VAChT) in the mouse forebrain. Executive function was assessed using a pairwise visual discrimination paradigm and the 5-choice serial reaction time task (5-CSRT). In the pairwise test, VAChT-deficient mice were able to learn, but were impaired in reversal learning, suggesting that these mice present cognitive inflexibility. Interestingly, VAChT-targeted mice took longer to reach criteria in the 5-CSRT. Although their performance was indistinguishable from that of control mice during low attentional demand, increased attentional demand revealed striking deficits in VAChT-deleted mice. Galantamine, a cholinesterase inhibitor used in Alzheimer's disease, significantly improved the performance of control mice, but not of VAChT-deficient mice on the 5-CSRT. In vivo magnetic resonance spectroscopy showed altered levels of two neurochemical markers of neuronal function, taurine and lactate, suggesting altered PFC metabolism in VAChT-deficient mice. The PFC of these mice displayed a drastic reduction in the splicing factor heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNPA2/B1), whose cholinergic-mediated reduction was previously demonstrated in Alzheimer's disease. Consequently, several key hnRNPA2/B1 target transcripts involved in neuronal function present changes in alternative splicing in VAChT-deficient mice, including pyruvate kinase M, a key enzyme involved in lactate metabolism. We propose that VAChT-targeted mice can be used to model and to dissect the neurochemical basis of executive abnormalities.

Alteration of the microRNA network during the progression of Alzheimer's disease.

An overview of miRNAs altered in Alzheimer's disease (AD) was established by profiling the hippocampus of a cohort of 41 late-onset AD (LOAD) patients and 23 controls, showing deregulation of 35 miRNAs. Profiling of miRNAs in the prefrontal cortex of a second independent cohort of 49 patients grouped by Braak stages revealed 41 deregulated miRNAs. We focused on miR-132-3p which is strongly altered in both brain areas. Downregulation of this miRNA occurs already at Braak stages III and IV, before loss of neuron-specific miRNAs. Next-generation sequencing confirmed a strong decrease of miR-132-3p and of three family-related miRNAs encoded by the same miRNA cluster on chromosome 17. Deregulation of miR-132-3p in AD brain appears to occur mainly in neurons displaying Tau hyper-phosphorylation. We provide evidence that miR-132-3p may contribute to disease progression through aberrant regulation of mRNA targets in the Tau network. The transcription factor (TF) FOXO1a appears to be a key target of miR-132-3p in this pathway.

Stereotactic injection of microRNA-expressing lentiviruses to the mouse hippocampus ca1 region and assessment of the behavioral outcome.

MicroRNAs (miRNAs) are small regulatory single-stranded RNA molecules around 22 nucleotides long that may each target numerous mRNA transcripts and dim an entire gene expression pathway by inducing destruction and/or inhibiting translation of these targets. Several miRNAs play key roles in maintaining neuronal structure and function and in higher-level brain functions, and methods are sought for manipulating their levels for exploring these functions. Here, we present a direct in vivo method for examining the cognitive consequences of enforced miRNAs excess in mice by stereotactic injection of miRNA-encoding virus particles. Specifically, the current protocol involves injection into the hippocampal CA1 region, which contributes to mammalian memory consolidation, learning, and stress responses, and offers a convenient injection site. The coordinates are measured according to the mouse bregma and virus perfusion is digitally controlled and kept very slow. After injection, the surgery wound is sealed and the animals recover. Lentiviruses encoding silencers of the corresponding mRNA targets serve to implicate the specific miRNA/target interaction responsible for the observed effect, with naïve mice, mice injected with saline and mice injected with "empty" lentivirus vectors as controls. One month post-injection, the animals are examined in the Morris Water Maze (MWM) for assessing their navigation learning and memory abilities. The MWM is a round tank filled with colored water with a small platform submerged 1 cm below the water surface. Steady visual cues around the tank allow for spatial navigation (sound and the earth's magnetic field may also assist the animals in navigating). Video camera monitoring enables measuring the route of swim and the time to find and amount the platform. The mouse is first taught that mounting the hidden platform offers an escape from the enforced swimming; it is then tested for using this escape and finally, the platform is removed and probe tests examine if the mouse remembers its previous location. Repeated tests over several consecutive days highlight improved performance of tested mice at shorter latencies to find and mount the platform, and as more direct routes to reach the platform or its location. Failure to show such improvement represents impaired learning and memory and/or anxiety, which may then be tested specifically (e.g. in the elevated plus maze). This approach enables validation of specific miRNAs and target transcripts in the studied cognitive and/or stress-related processes.

Statistically invalid classification of high throughput gene expression data.

Classification analysis based on high throughput data is a common feature in neuroscience and other fields of science, with a rapidly increasing impact on both basic biology and disease-related studies. The outcome of such classifications often serves to delineate novel biochemical mechanisms in health and disease states, identify new targets for therapeutic interference, and develop innovative diagnostic approaches. Given the importance of this type of studies, we screened 111 recently-published high-impact manuscripts involving classification analysis of gene expression, and found that 58 of them (53%) based their conclusions on a statistically invalid method which can lead to bias in a statistical sense (lower true classification accuracy then the reported classification accuracy). In this report we characterize the potential methodological error and its scope, investigate how it is influenced by different experimental parameters, and describe statistically valid methods for avoiding such classification mistakes.

Cholinergic-associated loss of hnRNP-A/B in Alzheimer's disease impairs cortical splicing and cognitive function in mice.

Genetic studies link inherited errors in RNA metabolism to familial neurodegenerative disease. Here, we report such errors and the underlying mechanism in sporadic Alzheimer's disease (AD). AD entorhinal cortices presented globally impaired exon exclusions and selective loss of the hnRNP A/B splicing factors. Supporting functional relevance, hnRNP A/B knockdown induced alternative splicing impairments and dendrite loss in primary neurons, and memory and electrocorticographic impairments in mice. Transgenic mice with disease-associated mutations in APP or Tau displayed no alterations in hnRNP A/B suggesting that its loss in AD is independent of Aß and Tau toxicity. However, cholinergic excitation increased hnRNP A/B levels while in vivo neurotoxin-mediated destruction of cholinergic neurons caused cortical AD-like decrease in hnRNP A/B and recapitulated the alternative splicing pattern of AD patients. Our findings present cholinergic-mediated hnRNP A/B loss and impaired RNA metabolism as important mechanisms involved in AD.

Similar cation channels mediate protection from cerebellar exitotoxicity by exercise and inheritance.

Exercise and inherited factors both affect recovery from stroke and head injury, but the underlying mechanisms and interconnections between them are yet unknown. Here, we report that similar cation channels mediate the protective effect of exercise and specific genetic background in a kainate injection model of cerebellar stroke. Microinjection to the cerebellum of the glutamatergic agonist, kainate, creates glutamatergic excito\xE2\x80\x90toxicity characteristic of focal stroke, head injury or alcoholism. Inherited protection and prior exercise were both accompanied by higher cerebellar expression levels of the Kir6.1 ATP-dependent potassium channel in adjacent Bergmann glia, and voltage-gated KVbeta2 and cyclic nucleotide-gated cation HCN1 channels in basket cells. Sedentary FVB/N and exercised C57BL/6 mice both expressed higher levels of these cation channels compared to sedentary C57BL/6 mice, and were both found to be less sensitive to glutamate toxicity. Moreover, blocking ATP-dependent potassium channels with Glibenclamide enhanced kainate-induced cell death in cerebellar slices from the resilient sedentary FVB/N mice. Furthermore, exercise increased the number of acetylcholinesterase-positive fibres in the molecular layer, reduced cerebellar cytokine levels and suppressed serum acetylcholinesterase activity, suggesting anti-inflammatory protection by enhanced cholinergic signalling. Our findings demonstrate for the first time that routine exercise and specific genetic backgrounds confer protection from cerebellar glutamatergic damages by similar molecular mechanisms, including elevated expression of cation channels. In addition, our findings highlight the involvement of the cholinergic anti-inflammatory pathway in insult-inducible cerebellar processes. These mechanisms are likely to play similar roles in other brain regions and injuries as well, opening new venues for targeted research efforts.

Threshold-independent meta-analysis of Alzheimer's disease transcriptomes shows progressive changes in hippocampal functions, epigenetics and microRNA regulation.

End-stage Alzheimer's disease (AD) involves drastic modifications in neuronal molecular and cellular processes, but little is known about the dynamics of these modifications during disease initiation and progression. Here, we report meta-analysis of 100 publicly available Microarray datasets using threshold-independent analysis. We found that different patients react to AD progression by variable single transcript alterations which however lead to similar changes in functional gene groups. Stratification by patients' cognitive deterioration pre`sented hippocampal-specific mRNA alterations which involved progressively changed gene categories and indicate changes in epigenetic state and microRNA profiles. In addition, datasets from laser-captured neurofibrillary tangles-free hippocampal neurons and transcript classification by cell types identified many of these changes in neurons. Intriguingly, we discovered that early-onset decline in alternative splicing, protein folding and transport transcripts occur concurrently with decreases in synaptic transmission, whereas at later stages these changes progressed into enhanced oxidative stress and inflammation. Our findings open new venues for identifying novel targets for intervention with AD progression.

Nicotinic stimulation induces Tristetraprolin over-production and attenuates inflammation in muscle.

Cholinergic signaling suppresses inflammation in blood and brain and attenuates apoptosis in other tissues, but whether it blocks inflammation in skeletal muscle under toxicant exposure, injuries and diseases remained unexplored. Here, we report nicotinic attenuation of inflammation and alteration of apoptotic protein expression pattern in murine muscle tissue and cultured myotubes, involving the RNA-binding protein, Tristetraprolin, and the anti-apoptotic protein, Mcl-1. In muscles and C2C12 myotubes, cholinergic excitation by exposure to nicotine or the organophosphorous pesticide, Paraoxon, induced Tristetraprolin overproduction while reducing pro-inflammatory transcripts such as IL-6, CXCL1 (KC) and CCL2 (MCP-1). Furthermore, nicotinic excitation under exposure to the bacterial endotoxin LPS attenuated over-expression of the CCL2 and suppressed the transcriptional activity of NF-ĸB and AP-1. Tristetraprolin was essential for this anti-inflammatory effect of nicotine in basal conditions. However, its knockdown also impaired the pro-inflammatory response to LPS. Finally, in vivo administration of Paraoxon or recombinant Acetylcholinesterase, leading respectively to either gain or loss of cholinergic signaling, modified muscle expression of key mRNA processing factors and several of their apoptosis-related targets. Specifically, cholinergic imbalances enhanced the kinase activators of the Serine-Arginine splicing kinases, Clk1 and Clk3. Moreover, Paraoxon raised the levels of the anti-apoptotic protein, Mcl-1, through a previously unrecognized polyadenylation site selection mechanism, producing longer, less stable Mcl-1 mRNA transcripts. Together, our findings demonstrate that in addition to activating muscle function, acetylcholine regulates muscle inflammation and cell survival, and point to Tristetraprolin and the choice of Mcl-1 mRNA polyadenylation sites as potential key players in muscle reactions to insults.

Unity vs. diversity of neuropathic pain mechanisms: Allodynia and hyperalgesia in rats selected for heritable predisposition to spontaneous pain.

Do contrasting neuropathic pain diagnoses share common pathophysiological mechanisms? Selective breeding was used to derive rat lines with a common genetic background but a striking difference in the degree of spontaneous pain behavior expressed in the neuroma model of neuropathic pain (HA rats (high autotomy) and LA rats (low autotomy)). The contrasting pain phenotype in these lines is attributable to allelic differences at a small number of genetic loci. Here we show that HA and LA rats also differ in their nocifensive response to applied stimuli in the Chung (spinal nerve ligation, SNL) model of neuropathic pain. This includes tactile allodynia and hyperalgesia, and heat allodynia. The degree of hypersensibility varied with sex, age at the time of nerve injury, and the extent of the nerve lesion. F1 crosses of HA and LA rats and inbred Lewis rats showed low levels of autotomy but variable levels of hypersensibility to applied stimuli. Results indicate that alleles which predispose to spontaneous neuropathic pain also predispose to stimulus-evoked pain (allodynia and hyperalgesia). This, in turn, suggests that despite contrasting etiology and behavioral endpoints, pain phenotype in the neuroma and the SNL models shares common pathophysiological mechanisms.