United States

Abstract Title
Hodgkin-Huxley Algorithm to Diagnose Channelopathies from Empirical Action Potentials

Background
Human channelopathies are diagnosed by symptom profile or genetic testing which fail to provide a mechanistic basis for patients’ experiences. This project aims to develop an algorithm that decomposes a patient’s electrophysiology data into clinically relevant values that would better focus treatment efforts. Our model system for variation in action potential pathology are garter snakes (Thamnophis spp.) that have evolved resistance to their neurotoxic prey (Taricha spp.). The toxin, tetrodotoxin, occludes the pore of voltage-gated sodium channels that conduct sensory and motor impulses. However, resistance mutations observed in snake skeletal muscle voltage-gated sodium channels significantly reduce channel function by limiting sodium conductance. Our model aims to reproduce that level of mechanistic detail from action potentials recorded in vivo directly in the target tissue rather than by complicated whole-cell electrophysiology techniques.

Methods
To observe sodium channel conductivity deficits, we collected action potentials from known healthy and pathological samples and then trained a model based on this dataset. We recorded from muscles carrying tetrodotoxin-sensitive sodium channels (NaV1.4⁺, N=9) from Thamnophis atratus as well as from snakes with mildly resistant channels (A1281P, N=11) suffering mild deficits. We then tailored the Hodgkin-Huxley model of the action potentials to fit skeletal muscle recordings. We deployed the model to decompose action potentials into distinct, molecularly-based mathematical parameters, including sodium channel conductance (GNa), sodium channel activation (m), and inactivation (h). We generated representative action potentials based on averages of each group. Then, R was used to fit a parameter set to each group to assess differences in molecularly-based parameters between groups (e.g. GNa).

Results
The model reproduced the expected negative correlation between sodium channel mutations and channel conductance (e.g. reduced GNa). Furthermore, it revealed significant decreases in two kinetic subparameters, m and h, providing an additional explanation of reduced conductance. These data validate our model against peer-reviewed research (del Carlo et al., 2024)

Conclusion
This model offers the first estimate of sodium channel function in native snake skeletal muscle. Our model produced results congruent with findings of peer-reviewed reports using gold-standard methods. However, the model currently lacks sufficient specificity to evaluate patient-level data. Ongoing work aims to expand this model as a diagnostic tool by training it on even more extreme snake channelopathies. With further refinement, this model could be a clinically useful tool for the differential diagnosis of human channelopathies, advancing personalized medicine for inherited disorders.

AMA Research Challenge 2024 

Hodgkin-Huxley Algorithm to Diagnose Channelopathies from Empirical Action Potentials

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The nicotinic acetylcholine receptor (nAChR) has been shown to play a functionally distinct role in the development of the adolescent brain through the modulation of neurotransmitter release across various neurodevelopmental milestones. CHRNA2 encodes for the α2 nicotinic acetylcholine receptor associated with CA1 oriens lacunosum moleculare GABAergic interneurons and is associated with learning and memory. Prior literature consistently supports the finding that chronic nicotine exposure during adolescence leads to an impairment in learning in adulthood. Previously, we found that adolescent male hypersensitive (CHRNA2 L9'S/L9'S) mice had impairments in learning and memory during a pre-exposure-dependent contextual fear conditioning task that could be rescued by low-dose nicotine exposure. In this study, female adolescent CHRNA2 L9'S/L9'S mice and wild-type (WT) littermate controls were exposed to saline or nicotine (0.09 mg/kg) using a hippocampus-dependent task of pre-exposure-dependent contextual fear conditioning. Alterations in freezing behavior—an indication of learning—were compared between CHRNA2 L9'S/L9'S and WT female adolescent mice as well as against adolescent male CHRNA2 L9'S/L9'S and WT mice. We found that nicotine-treated adolescent WT female mice had significantly greater freezing behavior than both saline-treated WT mice and nicotine-treated CHRNA2 L9'S/L9'S female mice. Nicotine-treated adolescent CHRNA2 L9'S/L9'S female mice did not have enhanced freezing behavior on context test (CT) day when compared with saline-treated CHRNA2 L9'S/L9'S female mice. These results indicate that hypersensitivity of the CHRNA2 gene in female adolescent mice produces deficits in nicotine-dependent learning and memory in the hippocampus. These results are contrasted with the results obtained in the previously published male data where we found that male hypersensitive (CHRNA2 L9'S/L9'S) mice had impairments in learning and memory that could be rescued by low-dose nicotine exposure. Thus, nicotine exposure mediates a sexually dimorphic pattern of learning and memory in WT and CHRNA2 L9'S/L9'S adolescent mice exposed to a pre-exposure-dependent contextual fear conditioning paradigm. At present, the mechanism driving sexual dimorphisms between nicotine-exposed adolescent CHRNA2 L9'S/L9'S mice is unknown. Further research must be conducted in both in-vitro and in-vivo studies examining the role of α2- expressing nAChR signaling mediating sex-dependent effects.

Hypersensitivity of the nicotinic acetylcholine receptor subunit (CHRNA2 L9’S) in female adolescent mice produces deficits in learning and memory that are not rescued by nicotine


Background 

Tumor cells in the tumor microenvironment (TME) are known to upregulate endoplasmic reticulum (ER) stress as a survival mechanism and ER stress transmission to the mitochondria is associated with tumorigenesis. We studied the mitochondrial unfolded protein response (mtUPR) associated Lon protease 1 (LONP1) in tumor cells undergoing ER stress. Our hypothesis is LONP1 inhibition during ER stress promotes immunogenic cell death (ICD) in melanoma cells.

Methods 

To simulate the TME, we used murine B16 melanoma cells that were treated with thapsigargin, a compound known to induce ER stress. Then, some cells were co-treated with both thapsigargin and CDDO-Me, a pharmacological inhibitor of LONP1. RT-PCR was used to assess potential targets associated with mt-UPR. TIMER is a database we used to assess the clinical significance of LONP1 in skin cutaneous melanoma patients. MitoProbe DIOC assay was used to assess mitochondrial membrane potential. Annexin V/PI assay was used to determine the proportion of apoptotic cells. Calreticulin exposure assay was used to determine the expression level of cell-surface calreticulin. CellTiter-Glo assay was used to determine both the intracellular and extracellular concentrations of ATP.

Results

RT-PCR showed that Lonp1 is expressed in response to ER stress. TIMER showed that expression of LONP1 is negatively correlated with the infiltration of cytotoxic CD8+ T cells, CD4+ T cells and activated NK cells. MitoProbe DIOC assay of co-treated cells showed the lowest mitochondrial membrane potential. Annexin V/PI assay of those cells showed an increase in apoptosis versus all other conditions. Calreticulin exposure assay of co-treated cells showed an increase in cell-surface calreticulin exposure versus all other conditions. CellTiterGlo assay showed both decreased intracellular ATP and increased extracellular ATP in cotreated cells only.

Conclusion

B16 melanoma cells undergoing ER stress in the absence of LONP1 function showed higher cell-surface calreticulin, higher extracellular ATP release, increased entry into late apoptosis and their mitochondrial membrane potential was significantly reduced. We conclude that inhibition of LONP1 in the presence of ER stress induced ICD in our cells, suggesting LONP1 as a potential target for cancer immunotherapy.

Inhibiting LONP1 Promotes Immunogenic Cell Death in Melanoma Cells Undergoing Endoplasmic Reticulum Stress

Background
Laser powder bed fusion (LPBF) is a 3D printing technique that rapidly and selectively melts metal powder via laser irradiation which then solidifies in the prescribed pattern before a new layer of powder is spread across the surface and the process repeats. LPBF is the most common 3D printing method used with stainless 316L alloys. It offers advantages in the biomedical field by improving mechanical properties such as fracture toughness and strength, enabling complex, patient-specific geometries and reducing material waste, crucial for applications like stents and implants. Nevertheless, consideration of corrosion behavior is crucial in assessing the longevity of these materials in aqueous environments. 

Methods
Various corrosion tests were conducted on LPBF-produced stainless steel alloys. These tests included polarization scans in acidic environments to assess corrosion resistance, mass loss testing in ferric chloride and sodium chloride solutions to measure susceptibility to localized corrosion, and galvanostatic etching in ammonium persulfate to identify microstructural sites prone to selective attack. Transmission electron microscopy was used to analyze local chemical composition differences at a microscale. Scanning electron microscopy and optical microscopy characterized the microstructure, focusing on features susceptible to localized corrosion.

Results
Corrosion testing showed lower rates for LPBF-produced material compared to conventionally processed stainless steel, suggesting enhanced corrosion resistance. However, post-test analysis revealed various localized corrosion forms, including dissolution through cellular structures formed during the rapid solidification inherent to LPBF. Transmission electron microscopy uncovered local variations in elements critical to corrosion resistance like chromium and molybdenum, impacting corrosion behavior. These findings emphasize the complexity of corrosion mechanisms in LPBF materials and the importance of detailed microstructural analysis.

Conclusion
The study identified lower corrosion rates in LPBF-produced stainless steel alloys but also highlighted localized corrosion pathways through cellular structures, pores formed due to inadequate metal fusion, and other microstructural features inherent to the LPBF process. Advanced microscopy techniques revealed elemental variations influencing corrosion behavior. Future research should explore diverse environments, long-term corrosion performance, and predictive models linking microstructure to corrosion behavior. Addressing these challenges is vital for developing corrosion-resistant materials for biomedical applications and enhancing device performance in clinical settings.

Investigating Corrosion Behavior in 3D Printed Laser Powder Bed Fusion Stainless Steel 316L Alloys - Insights into Material Behavior Relevant for Biomedical Applications

P21-activated kinase 1 (PAK1) is a serine/threonine protein kinase that plays a crucial role in cardiomyocyte survival in response to various stresses. However, the underlying cardioprotective mechanisms of PAK1 remain unclear. Autophagy is a lysosome degradation pathway essential for cellular homeostasis. In the present study, we showed the ability of PAK1 to regulate autophagy in cardiomyocytes and identified p62 as a potential downstream mediator. PAK1 is silenced in H9c2 cardiac myoblasts by siRNA or overexpressed by adenovirus. Autophagy flux was determined by LC3-II protein levels and a fluorescent autophagy reporter with and without the lysosomal protease inhibitors Pepstatin A (pepA) and Aloxistatin (E64d). Downregulation of PAK1 reduced LC3-II levels in whole cell lysates, which was not affected by pepA and E64d, suggesting that PAK1 knockdown reduced autophagy flux. Conversely, PAK1 overexpression increased LC3-II levels, which were further elevated by pepA and E64d, suggesting that PAK1 is sufficient to promote autophagy. Interestingly, the protein expression of p62, a ubiquitin-binding autophagy adaptor, was increased by PAK1 overexpression and reduced by PAK1 knockdown. Importantly, overexpression of p62 rescued the defective autophagy flux in the absence of PAK1, suggesting that p62 may mediate PAK1-dependent autophagy. RT-qPCR assay showed that the p62 mRNA levels were unchanged by either overexpression or knockdown of PAK1 in vitro and in vivo. The cells were treated with the protein synthesis inhibitor cycloheximide (CHX) and p62 protein levels were measured over time in both PAK1-silencing and overexpressing conditions. The results showed that p62 degradation was mitigated by PAK1 overexpression and accelerated by PAK1 knockdown. Together, these results suggest that PAK1 is sufficient and necessary to maintain autophagy in cardiomyocytes, which is likely mediated by its ability to affect p62 protein stability.

PAK1 regulates cardiomyocyte autophagy through p62

Kv7 channels are potassium ion channels expressed in the nervous system. Kv7 channels function to control the excitability of neurons by reducing the rate of action potential firing. The Kv7.2 channel has been shown to play a particularly crucial role in neurodevelopment. Five different KCNQ genes encode Kv7 subunits, which form a tetrameric Kv7 channel. Loss of function mutations in the KCNQ2 gene have been shown to result in neurologic dysfunction, including neonatal epilepsy. Therefore, regulation of Kv7.2 is essential for normal neuronal function. Calmodulin (CaM) is a calcium (Ca2+)-binding protein shown to interact with the C-terminus of Kv7.2, particularly with its intracellular regulatory domain. This region contains several hotspots for pathological mutations, suggesting their importance in regulating the function of Kv7.2. The regulatory domain of KCNQ2 is composed of two regions of importance to this study: the A helix (Q2A) and B helix (Q2B) (Fig. 1 & 2). CaM binds to each Kv7 subunit by interacting with Q2A and Q2B. The amino acid sequence of Q2B contains phosphorylation sites at serines S520 and S527 which could potentially contribute to the regulation of Kv7.2 function. This study aims to characterize the Ca2+-dependent binding interactions between CaM and the regulatory domain of Kv7.2. It is also investigating whether phosphorylation of Q2B interferes with CaM interactions and how CaM modifies its binding to the regulatory domain. Binding interactions were studied by electrophoretic mobility shift assay (EMSA) and isothermal calorimetry (ITC). Results from the ITC show that CaM has a higher affinity for Q2B (Kd = 0.243 μM) than Q2A (Kd = 5.27 μM). Compared to the binding affinity between CaM and an extended Q2B sequence (Kd = 0.05 μM), the binding affinity is 42x lower when Q2B is phosphorylated at S520 (Kd = 2.1 μM) and only 5x lower when phosphorylated at S527 (Kd = 0.25 μM). EMSA shows a degree of CaM binding to Q2B phosphorylated at S527 but demonstrates no binding when phosphorylated at S520. These findings indicate that Q2B phosphorylation at S520 blocks CaM interactions in the presence of Ca2+.

Phosphorylation of Kv7.2 Potassium Channel's Regulatory Domain Alters Binding Interactions with Calmodulin

Background
An individual’s healthcare costs can provide an objective measure of overall morbidity. Quantifying the impact of genetic variation on healthcare costs can inform public health policies and help identify genetic determinants of mortality. 

Methods
We estimated inpatient, outpatient, primary care and prescription-related costs, from the perspective of healthcare systems, for a total of 1.3 million individuals across 8 different healthcare systems. The median per-patient annual healthcare cost (expressed in 2019 prices) had substantial range across countries, from €483/year in the Estonian Biobank to €458,455/year in the Mass General Brigham Biobank. 

A GWAS meta-analysis identified 27 and 106 significant, independent loci for inpatient and medication costs, respectively. Effects were overall modest, the most significant inpatient signal (rs6938239 near ILRUN) was associated with ~1% increased cost per year, per allele. Following stratified analyses, 33% of inpatient loci had sex-specific effects while 22% had an age-specific effect. E.g. homozygous carriers of variant rs62236881 in the 3’ UTR of ZNRF3 had 9% higher costs in females compared with 4% in males, potentially due to an increased risk of breast cancer.

Results
Despite differences in tariff structure and cost reimbursement across healthcare systems, we observed strong genetic correlations between inpatient studies (median rg=0.88) and 95% of significant loci did not show heterogeneity across studies. PheWAS indicated that significant loci may impact healthcare costs via either common risk factors (e.g. BMI) or chronic diseases risk (e.g. asthma, depression).

Leveraging whole-exome sequencing data from the UK Biobank (UKB), we estimated the cost for putative loss of function (pLOF) burdens for 83 clinically relevant genes. The largest effects were observed for BRCA1/2, MSH2 and APC. Annual inpatient costs were ~1.8 times higher in individuals carrying one pLOF in BRCA2 than in non-carriers. We estimated that the total annual inpatient costs attributed to BRCA2 pLOF mutations amongst those aged 40-69 in the UK would be about €26 million.

Polygenic scores (PGS) for various risk factors and diseases were linked to higher healthcare costs. In UKB, the top 10% with the highest BMI PGS had a median annual cost of €414, compared to €329 for those in the middle 40-60%. Using polygenic weights from GenCost data, PGS for ~11,000 individuals in Genomics England showed that the top 10% incurred ~€250 more than those in the middle 40-60%.

Conclusion
Genetic variation across individuals influences healthcare expenditure. Our findings are applicable across various healthcare systems and help better understanding global morbidity.

Quantifying the impact of genetic variation on healthcare cost across 1.3 million individuals from 11 studies and 8 countries: The GenCost Consortium

Background: Hirschsprung disease is a congenital neurocristopathy that affects 1 in 5000 newborns. The disease involves fetal gut aganglionosis due to the defective migration and differentiation of neural crest cells in the intestines. The condition is associated with intractable constipation, megacolon ileus and the need for colectomy in childhood. While it was previously known that gestational exposures such as maternal vitamin A deficiency, ibuprofen intake, and BP-3 sunscreen use had implications on the development of Hirschsprung, we found that these variables share effects common to the RET proto-oncogene pathway.
Methods: An initial systematic review was done on the NIH library, PubMed and Scorpus databases to evaluate existing literature on maternal gestational exposures tied to Hirschsprung disease. Search queries involved keywords “Hirschsprung disease”, “maternal exposure” and “developmental pathway”. Inclusion criteria for accepted studies were: peer reviewed status, sample size greater than twenty, human models, discussion of developmental mechanisms, and published within the past twenty years. Based on these studies, we categorized the maternal exposure variables and further reviewed the specific biologic pathways impacted. From this list of pathways, the mechanism common between the highest number of categories was selected for this report. 
Results: This study explores how maternal exposures to vitamin A deficiency, BP-3, and ibuprofen increase the risk of Hirschsprung disease by affecting the RET proto-oncogene, which is essential for neural crest cell migration and implicated in at least 20% of all Hirschsprung cases. Retinoic acid, the active form of vitamin A, upregulates RET transcription. As a result, low vitamin A levels can downregulate RET and impair neural crest cell migration. Benzophenone-3 (BP-3), a sunscreen ingredient, reduces RET levels by disrupting fetal enteric neuron development and neural crest cell migration via the SLIT2/ROBO1-miR-218-RET/PLAG1 pathway. While ibuprofen does not directly affect RET, it may affect RET signaling by increasing acetylcholinesterase (AchE) activity, as suggested by studies showing co-expression of RET and choline acetyltransferase in enteric ganglia.
Conclusion: 
Our study found that vitamin A deficiency, BP-3, and ibuprofen increase the risk of Hirschsprung disease via different pathways, with the RET proto-oncogene being the most commonly implicated gene. Vitamin A deficiency and BP-3 exposure both affect the RET signaling pathway, while ibuprofen appears to indirectly increase risk through a separate mechanism. These findings highlight the need to monitor and mitigate maternal exposures to reduce Hirschsprung disease risk and suggest the RET proto-oncogene as a potential target for treatment and prevention.



RET Proto-Oncogene Links Gestational Exposures Implicated in Hirschsprung’s Development: A Review

S. Hamza Naqvi ‡ , Alexandra Yokomizo ‡ , Jonmark Dolendo ‡ , David C. Swinney ¶,§ , Brad A. Haubrich ‡


Background: Human African trypanosomiasis (HAT, also called sleeping sickness) is
caused by the parasite Trypanosoma brucei. HAT is one of three diseases caused by
kinetoplastid protozoa (the TriTryp diseases), alongside Chagas disease and
leishmaniasis. HAT and the other TriTryp diseases are neglected, meaning endemic
populations have less resources, and there is consequently little funding or commercial interest in discovery and development of new drugs. One strategy to expedite drug discovery is to test known compounds for antiparasitic activity. Towards this goal, we have developed in vitro assays for an essential T. brucei MAP kinase and screened a small library of known human protein kinase inhibitors (PKIs).

Methods: PKIs were purchased from chemical and pharmaceutical suppliers, and
TbMAPK6 was produced by Biozilla (Dallas TX). Enzyme assays, in the presence or
absence of PKIs, were run at 37°C and stopped with heat. Reactions were monitored
with ADP-Glo assay kits (Promega, Madison WI). Enzymatic and inhibition data was
analyzed with GraphPad Prism (New York, NY), and protein alignments were conducted with UniProt.

Results: TbMAPK6 phosphorylated myelin basic protein with Michaelis-Menten kinetics, and its activity was linear with respect to both time and enzyme concentration. Kms for MBP and ATP were found to be 47 μM and 7.7 μM, respectively. Pan-kinase inhibitor staurosporine had an IC 50 of 120 nM. Other PKIs tested varied in potency by orders of magnitude. TbMAPK6 aligns with human ERK7/8 with 43.5 percent identity and to several other putative pathogenic MAP kinases at varying identities. PKIs screened included compounds that were not developed for ERK7/8. 

Conclusion:  The differential inhibition among PKIs underscores the potential for future drug discovery in the area of Neglected Tropical Diseases. Furthermore the second key finding was through our percent Identities of TbMAPK6 with MAP kinases from other parasites. This finding will allow additional potential of repurposing for other neglected diseases with higher epidemiological burdens than HAT. Next steps will be to expand our PKI library against TbMAPK6 and expand screening to other pathogenic MAP kinases.

RoadMAP for New TriTryp Therapeutics: Screening of a Trypanosomal
MAP Kinase

Background
The spaceflight environment imposes harmful stressors such as microgravity and ionizing radiation on the human body. The eye is especially susceptible to damage from these stressors due to their effects on mitochondrial function. These stressors result in mitochondrial DNA damage, oxidative stress, impaired oxidative metabolism, and overall decreased mitochondrial function. Mitochondrial DNA acquires mutations more readily than nuclear DNA and has weaker genetic repair mechanisms. The result of these insults is the development of spaceflight-associated ocular pathologies such as Light Flashes (LF), Cataracts, and Spaceflight Associated Neuro-Ocular Syndrome (SANS). Mitochondrial dysfunction is becoming more apparent as a causative factor of these conditions. Therefore, a greater understanding of the pathogenesis is necessary to develop effective countermeasures to preserve ocular health in astronauts as they embark on longer missions.

Methods
A literature review was conducted using online databases such as PubMed and Science Direct. The reference articles were analyzed to develop a narrative.

Results
Conditions that impact astronauts in spaceflight have associations with terrestrial conditions but exhibit key distinctions, pointing to mitochondrial involvement in pathogenesis. Astronauts are more likely to develop cortical cataracts, which occur in the periphery of the lens where mitochondria are abundant. The most common type of cataract on Earth is the nuclear cataract, occurring in the center of the lens where there are no mitochondria. The retina has many mitochondria, and the damage imparted by spaceflight manifests as SANS, characterized by optic disc edema, globe flattening, choroidal retinal folds, and hyperopic refractive error shifts. Recent data point to mitochondrial dysfunction in photoreceptors and supporting structures due to radiation, oxidative stress, and vitamin/metabolic deficiencies as a cause of this unique condition.

Conclusion
Spaceflight damages mitochondria within the eye, contributing to development of ocular diseases such as cataracts and SANS. Astronauts develop cortical cataracts due to the distribution of lens mitochondria and susceptibility to oxidative damage and ionizing radiation. Systemic oxidative stress also contributes to the development, thus antioxidant therapy may be a treatment. Cataracts and SANS development may be connected, as shown by the development of cotton-wool spots experienced by astronauts. Future missions to the Moon and Mars will exacerbate the negative effects of spaceflight experienced in low earth orbit and present novel stressors. Advanced medical care will not be readily accessible during long-duration missions. These demands warrant a greater understanding of the pathogenesis of ocular conditions associated with spaceflight for the development of appropriate countermeasures.


Spaceflight Stressors Impact on Mitochondrial Function and the Development of Ocular Pathology

Background
Despite a decade-long reduction in opioid prescriptions, opioid misuse remains a major health crisis causing most drug overdose fatalities. Meanwhile, gabapentin (Neurontin®) prescriptions have doubled. Gabapentin is present in over 10% of drug overdose fatalities, 90% of which involve opioids. Although reports show gabapentin is prescribed off-label for opioid withdrawal, empirical evidence is lacking regarding its efficacy in mitigating opioid withdrawal symptoms or modulating opioid dependence. It is hypothesized that gabapentin attenuates expression of opioid withdrawal.

Methods
Opioid dependence was established in male Sprague-Dawley rats administered escalating morphine doses (10-40 mg/kg) twice daily for four days. Morphine withdrawal was measured using food-maintained responding, withdrawal signs, and body weight loss in six rats trained to lever press for a sucrose pellet (fixed-ratio 5-response schedule of reinforcement). The operant session comprised five cycles: a 5-minute post-saline timeout and a 10-minute response period. Gabapentin doses (3.2-320 mg/kg) were cumulatively administered for testing and dependence was maintained with a morphine dose (40 mg/kg) after daily operant experiments.

In a separate experiment, seven groups of rats (N=8/group; Groups A - G) were observed for 14 somatic withdrawal signs, and changes in body weight. Opioid dependence was established in Groups A, B, D, and F with morphine doses (10-40 mg/kg), while Group C, E, and G received saline. Subsequently, Group A, the morphine-dependent control group, received saline injections at 7 AM and 5 PM, whereas the rest of the groups received gabapentin (32, 100, or 320 mg/kg) at 7 AM and saline at 5 PM daily. Body weight and rotarod performance were used to quantify effects following morphine discontinuation. 

Results
Following opioid dependence, response rates stabilized just below 35% of baseline, and body weight loss was 20 g greater at 7 AM (14 hours after administration of saline) than at 5PM (10 hours after administration of 40 mg/kg morphine). Morphine and gabapentin significantly increased rate of responding. Only 32 mg/kg gabapentin significantly attenuated body weight loss and withdrawal signs on days 1 and 2. Enhancement of rotarod performance and hypothermia were significantly attenuated by 320 mg/kg gabapentin. No dose of gabapentin significantly attenuated mechanical allodynia. 

Conclusion
These results fail to support the view that gabapentin attenuates opioid withdrawal in male rats. Further research is needed to clarify its role in mitigating opioid withdrawal and dependence, considering potential influences from other factors or perceived benefits.


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Hypersensitivity of the nicotinic acetylcholine receptor subunit (CHRNA2 L9’S) in female adolescent mice produces deficits in learning and memory that are not rescued by nicotine

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