Visual snow syndrome is a persistent visual disturbance characterized by rapid flickering dots in the entire visual field. Hypothesized to arise from reduced inhibition of sensory cortex, visual snow has recently been linked to potentiation (enhancement) of the P100, an event-related potential (ERP) component associated with early visual processing. Here, we investigate whether this potentiation in visual snow is specific to visual responses, by comparing ERPs linked to early, bottom-up perceptual versus late, top-down cognitive processes. Specifically, we examined two components, the N170 and P300, associated respectively with rapid face categorization and attentional orienting towards targets. We predicted that if visual snow predominantly reflects diminished inhibition of perceptual areas, there should be stronger potentiation for the earlier perceptual N170 component. ERPs associated with the N170 (Face > House) and P300 (Target > Nontarget) were recorded in a 22 year-old male with a 2-year history of visual snow symptoms and a set of age- and gender-matched controls. Although N170 and P300 responses in all participants showed appropriate face- and target-selectivity, respectively, the visual snow patient demonstrated consistent potentiation relative to controls, particularly for the early N170 response. Bootstrapped estimates of mean amplitude computed within participants similarly revealed larger and more variable ERP amplitudes in the visual snow patient, especially for the N170 component. These results support an early perceptual locus of ERP potentiation in visual snow, further supporting the idea that this condition arises from diminished inhibition of sensory cortices.

During neuronal development, the axonal and dendritic projections are polarized and oriented along specific body axis. To further explore the molecular basis of neuritogenesis in vivo, we used the nematode Caenorhabditis elegans as a developmental model and performed a forward genetic screen to identify genes that specify the polarity of neurite outgrowth. We examined the VC4 and VC5 neurons, members of the six VC motor neurons using the Pcat-1

Spontaneously active frontal cortex and spinal cord networks grown on microelectrode arrays were used to study effects of D-methylphenidate. These central nervous system tissues have relatively low concentrations of dopaminergic and noradrenergic neurons compared to the richly populated loci, yet exhibit similar neurophysiological responses to methylphenidate. The spontaneous spike activity of both tissues was inhibited in a concentration-dependent manner by serial additions of 1-500 µM methylphenidate. Methylphenidate is non-toxic as spike inhibition was recovered following washes. The average concentrations for 50% spike rate inhibition (IC50 ± SD) were 118 ± 52 (n= 6) and 57 ± 43 (n = 11) for frontal cortex and spinal cord networks, respectively. A 3 hour exposure of a network to 1 mM methylphenidate was nontoxic. The effective concentrations described in this study are within the therapeutic dosage range. Therefore, the platform may be used for further investigations of drug mechanisms.

Investigation of the neural correlates of the self has implicated a network of brain regions including the medial prefrontal cortex (MPFC), posterior cingulate (PCC), precuneus (pC), and inferior parietal lobule (IPL). At the same time, recent neuroimaging work has identified the Default Mode Network (DMN), a network of brain regions that are highly active at `rest' (without an active cognitive task). While the functional significance of the DMN remains unknown, converging evidence suggests that the DMN might be critical for self-referential processing (e.g., introspection). In this dissertation, I tested this hypothesis using a lesion approach. In the first experiment, I examined the critical role of the DMN hubs (MPFC, IPL) in autobiographical memory (AM) retrieval. I predicted that if the DMN hubs were critical for AM, then lesions to either the MPFC or IPL should result in AM retrieval impairments. I tested this prediction using the Iowa Autobiographical Memory Questionnaire (IAMQ), a questionnaire that assessed retrograde AM retrieval. In support of the prediction, lesions to the MPFC and IPL were associated with significant AM retrieval impairments. While not predicted, AM retrieval deficits were also associated with lesions in medial and lateral temporal cortices, regions also considered part of the DMN. In the second experiment, I tested the critical role of the DMN hubs in the self-reference effect (SRE), a well-known memory advantage conferred by self-related processing. I predicted that if the DMN hubs were critical for the SRE, then damage to the MPFC or IPL should diminish the effect. I used a standard personality trait judgment paradigm to test this prediction. In partial support of the prediction, I found that damage to the MPFC abolished the SRE with a "self" specific deficit. While IPL damage was associated with a diminished SRE, the effects were not significant. In the third experiment, I tested the hypothesis that the DMN is critical for accurate knowledge of one's personality. I predicted that if the DMN hubs are critical for accurate self-knowledge, then damage to either the MPFC or IPL should be associated with less accurate personality reports. In partial support of the prediction, MPFC and IPL groups demonstrated less accurate personality ratings. However, performance for all lesion groups was comparable and not significantly different from healthy subjects. In the fourth experiment, I sought to test the hypothesis that the DMN is critical for mind wandering (MW). I predicted that if the DMN hubs are critical for MW, then damage to the MPFC and IPL should result in decreased MW. To test this prediction, I used two approaches: 1) an experience sampling method (Sustained Attention to Response Task), and 2) a self-report measure (Imaginal Processes Inventory scale of MW). Contrary to my prediction, IPL lesions were associated with increased MW on the SART. By contrast, in support of the prediction, both MPFC and IPL lesions were associated with significant self-reported decreases in MW. Together, these experiments provide some evidence to support the hypothesis that the DMN is critical for self-referential processing. Future work might investigate the impact of DMN lesions on other self-processes (e.g., self-agency).

Chronic pain is a significant healthcare problem. It is disabling and diminishes quality of life. Opioids, such as morphine, remain a primary pharmacologic management for chronic pain. Opioids act at mu opioid receptors (MOPr) in the rostroventral medial medulla (RVM) to produce their analgesic effect. The RVM is a critical relay in pain inhibitory and facilitatory pathways of pain modulation. Furthermore, chronic inflammatory pain, produced by CFA hindpaw injection, leads to adaptive changes in the RVM that change the balance of these pathways and increase the potency of opioids. MOPr are known to produce their effects via Gi/o proteins. Pretreatment of several pain modulatory regions with pertussis toxin (PTX) effectively attenuates the antinociceptive effects of MOPr agonists, such as DAMGO. In the RVM, PTX effectively reduced DAMGO stimulated GTPãS binding in uninjured rats. However, despite their effective inactivation of Gi/o proteins, PTX did not diminish the antinociceptive effects of DAMGO in the RVM of uninjured rats. In contrast, in rats with a chronic inflammatory injury, PTX completely abolished the antinociceptive effects of DAMGO. These results suggest a transition from Gi/o independent to Gi/o dependent mechanisms following CFA treatment. In addition, the anti-hyperalgesic effects of DAMGO were not inhibited by PTX, suggesting that DAMGO produces anti-hyperalgesia and antinociception by different mechanisms. In the RVM, MOPr are present both postsynaptically and presynaptically. Postsynaptic MOPr are thought to produce antinociception by activating GIRK channels, resulting in hyperpolarization and inhibition of pain facilitatory neurons. Indeed, inhibition of GIRK channels in the RVM, via microinjection of tertiapin-Q, attenuated the antinociceptive effects of DAMGO in uninjured rats, providing the first behavioral evidence that MOPr agonists produce analgesia via this proposed mechanism. Interestingly, however, tertiapin-Q did not block the anti-hyperalgesic effects of DAMGO, nor did it diminish the antinociceptive effects of DAMGO in the contralateral hindpaw of CFA treated rats. Furthermore, these differential effects of tertiapin-Q in the uninjured and injured rats are not the result of transcriptional down regulation of GIRK channels in the RVM. Finally, tertiapin-Q alone in the RVM produced a modest antinociception in uninjured rats, providing the first evidence of constitutive GIRK channel activity in the RVM and demonstrating a role for these in pain modulation. Presynaptic MOPr are thought to produce antinociception by decreasing GABA release onto pain inhibitory neurons. Indeed, microdialysis studies demonstrated that levels of GABA release were decreased in response to DAMGO perfused into the RVM, as well as to high potassium after perfusion of DAMGO. However, they were not decreased in rats after CFA treatment. This suggests that chronic inflammatory injury alters the presynaptic actions of MOPr agonists in the RVM. Interestingly, levels of GLU release where not altered by DAMGO in uninjured or injured rats. Moreover, basal levels of GLU and GABA were also unaltered by CFA treatment. In conclusion, although MOPr mediate their antinociceptive effects in other pain modulatory regions via Gi/o proteins, this is not the case in the RVM during an uninjured state. However, MOPr-induced antinociception transitions from Gi/o independent to Gi/o dependent mechanisms after CFA treatment. Additionally, these results support both the presynaptic and the postsynaptic postulates by which MOPr agonists are thought to produce their analgesic effects. However, although CFA treatment alters the activity of neurons in the RVM and promotes changes that result in an enhanced anti-hyperalgesic and antinociceptive response to DAMGO in the RVM, neither the postsynaptic nor the presynaptic mechanism, in isolation, seem to account for this enhancement.

It is a well-known phenomenon that stress can lead to hippocampal damage and a subsequent decline in anterograde memory. We are now learning that stress may also damage the prefrontal cortex, a brain region involved in important cognitive abilities such as judgment and decision making. Notably, several of the brain regions vulnerable to increased levels of stress (i.e., hippocampus and prefrontal cortex), are also known to undergo disproportionate decline during normal aging. To date, surprisingly very little research has examined the effects of stress on the prefrontal cortex and decision-making preferences in the elderly. In order to address these gaps in the literature, the aim of the current study was to investigate how stress impacts the prefrontal cortex and decision-making preferences in a healthy older adult sample. The first aim was to investigate the impact of acute stress on decision making in older adults. It was hypothesized that typical age-related changes in decision making would be amplified in older adults when subjected to an acute stressor, the Trier Social Stress Test (TSST). Decision-making preferences were measured by tasks that assess decision making under risk (via the Cups Task), decision making under ambiguity (via the Ellsberg Task), and temporal discounting (via the Intertemporal Choice Task). In partial support of this prediction, stress induced by the TSST affected decision making under risk and temporal discounting in older adults. After the TSST, older adults older adults displayed decreased risk-seeking when presented with a gain. Moreover, after the TSST, older adults displayed increased temporal discounting. That is, they had a decreased preference for later but larger gains (vs. smaller but sooner gains) and an increased preference for later but larger losses (vs. smaller but sooner losses). Moreover, changes in decision making varied depending on whether or not participants showed a physiological response, as measured by cortisol, to the TSST. The second aim was to investigate the relationship between chronic stress and decision making in older adults. It was hypothesized that typical age-related changes in decision making would be amplified in older adults that evidenced higher levels of chronic stress. Chronic stress was measured by: 1) chronic stress self-report questionnaires; 2) a semi-structured chronic stress clinical interview; 3) 24-hour urinary free cortisol; and 4) diurnal salivary cortisol. Decision-making preferences were measured by tasks assessing decision making under risk (via the Cups Task), decision making under ambiguity (via the Ellsberg Task), and temporal discounting (via the Intertemporal Choice Task). Overall, I did not find strong support for the prediction that decision making and chronic stress are related. The third aim was to investigate the relationship between chronic stress and the integrity of the prefrontal cortex in older adults. It was hypothesized that older adults who evidenced more chronic stress would have decreased volumes of the prefrontal cortex, as measured by structural magnetic resonance imaging (MRI). Chronic stress was measured by: 1) chronic stress self-report questionnaires; 2) a semi-structured chronic stress clinical interview; 3) 24-hour urinary free cortisol and 4) diurnal salivary cortisol. As predicted, perceived stress was negatively correlated with prefrontal cortex (PFC) volumes. Contrary to what was predicted, increased urinary free cortisol, diurnal salivary cortisol and positive life stressors (LES-Positive) were positively correlated with PFC volumes. Together these experiments provide some evidence to support the hypothesis that stress, both acute and chronic, impacts the prefrontal cortex and decision making among healthy older adults.

Les lysosomes contiennent environ soixante hydrolases différentes, qui peuvent dégrader une grande variété de macromolécules. L’activité de ces enzymes est dépendante du pH, maintenu dans les lysosomes entre 4.5 et 5.0 par une pompe à protons : la v-ATPase. Les produits de dégradation sont recyclés dans le cytoplasme par des transporteurs actifs secondaires de la membrane des lysosomes.Les maladies de surcharges lysosomales sont causées par des mutations de gènes codant pour des protéines lysosomales, souvent des enzymes. Elles sont caractérisées par un engorgement des lysosomes avec des agrégats ou des cristaux. Les symptômes associés à ces maladies sont variés, mais la moitié d’entre elles induisent des défauts neurologiques. L’étude de ces maladies a permis d’élucider la fonction de nombreuses enzymes, mais la connaissance des transporteurs lysosomaux reste parcellaire. Peu de ces transporteurs sont ainsi caractérisés au niveau moléculaire.Je me suis intéressé à deux gènes dont la mutation provoque une maladie de surcharge particulière : CLN3 et CLN7. Leur mutation provoque des céroïdes lipofuscinoses neuronales, des maladies de surcharge lysosomales caractérisées par une neurodégénérescence précoce et par l’accumulation dans les lysosomes d’un pigment autofluorescent, la lipofuscine. La mutation de 14 gènes différents cause une céroïde lipofuscinose neuronale. J’ai étudié CLN3 et CLN7 car ils codaient pour des protéines membranaires du lysosome, qui pourraient donc être des transporteurs.Sur CLN7, j’ai effectué des tests de transport en utilisant les acides aminés comme substrats potentiels, sans résultats probants. Concernant CLN3, le contenu métabolique de lysosomes a été étudié par spectrométrie de masse dans des souris WT ou de souris où le gène CLN3 était déficient. Les lysosomes des cellules déficientes contenaient moins de produits de la protéolyse, ce qui suggérait que CLN3 était important pour la protéolyse lysosomale. Cela a été confirmé par des mesures plus directes sur des neurones et des fibroblastes primaires, et sur des fibroblastes immortalisés. Ces résultats pourraient aider à comprendre les premières étapes de la physiopathologie dans les cellules où des gènes CLN sont déficients.Pour accroître le nombre de transporteurs lysosomaux potentiels, j’ai participé à la finalisation d’une étude par protéomique de la membrane lysosomale. Elle a révélé 46 potentiels transporteurs de fonction encore inconnue. Dans cette liste, j’ai étudié TMEM104, SPINSTER, MFSD1, SLC37A2, TTYH3 et SNAT7. Pour ce faire, j’ai d’abord muté les motifs d’adressage de ces protéines pour les rediriger, lors de leur synthèse, vers la membrane plasmique, afin de faciliter leur étude. Aucune fonction claire n’a pu être identifiée par cette approche.SNAT7 appartenait cependant à une famille de transporteurs de glutamine, ce qui était suffisamment encourageant pour envisager d’autres approches. Sa fonction a ainsi été étudiée en développement un nouveau test indirect basé sur la détection d’une surcharge artificielle des lysosomes en acides aminés. Un test fonctionnel plus direct a ensuite été mis au point sur des fractions enrichies en lysosomes en utilisant des acides aminés radiomarqués. Ces deux tests ont montré que SNAT7 était un transporteur spécifique de l’asparagine et de la glutamine.J’ai enfin étudié l’hypothèse suggérant que SNAT7 pourrait jouer dans la nutrition de cellules cancéreuses. En effet, certaines utilisent la glutamine comme nutriment principal à la place du glucose ; mais les apports sanguins en glutamine, dans les tumeurs, sont parfois insuffisants. La glutamine est donc obtenue par macropinocytose de protéines extracellulaires et dégradation lysosomale de ces protéines, avant un recyclage vers le cytoplasme. J’ai montré qu’en l’absence de SNAT7, ce phénomène était bloqué. SNAT7 est donc une cible thérapeutique intéressante pour tenter de bloquer l’approvisionnement des cellules cancéreuses en glutamine. / Within lysosomes, about sixty different hydrolases degrade macromolecules. This degradation is dependent on the acidity of the lysosomal lumen, which pH ranges between 4.5 and 5.0. The lysosomal pH is maintained by the v-ATPase, a proton pump. Lysosomal degradation generates catabolites, which can be recycled to cytosol by secondary active transporters: lysosomal transporters.The dysfunction of lysosomal proteins leads to lysosomal storage disorders (LSDs), rare inherited metabolic diseases characterised by accumulation of material inside lysosomes. Depending on the mutated gene, symptoms of LSDs vary greatly, although about half of LSD patients display some kind of neurodegenerative symptoms. Studying the physiopathology of LSDs has led to a good understanding of the function of lysosomal enzymes, but the knowledge of lysosomal transporters remain poor, since only a few LSDs has been shown to be linked with a mutation in a lysosomal transporter gene.I focused on two proteins which dysfunction causes a special type of LSDs: CLN3 and CLN7. Mutations in CLN3 and CLN7 cause neuronal ceroid lipofuscinoses (NCLs), a special type of LSD which has mostly neurodegenerative symptoms and which is characterized by the accumulation of a specific pigment inside lysosomes: lipofuscin. There are fourteen NCL genes, but CLN3 and CLN7 are the two only proteins of the family which are resident proteins of the lysosomal membrane, suggesting they might be transporters.Amino acids were screened as possible substrates for CLN7, but none could be shown to be transported. For CLN3, the content in metabolites of lysosomes from Cln3-deficient mice and from WT mice were compared by mass spectrometry, revealing a specific decrease in the amount of catabolites of proteins in lysosomes from Cln3-deficient mice. This suggested a lack of lysosomal proteolysis, which was checked in neurons, in primary fibroblasts and in immortalized fibroblasts. These results suggested that CLN proteins could take part to a metabolic pathway important for lysosomal proteolysis and, more generally, for neuronal health. These results could help improve the understanding of the early steps of NCL physiopathology.To extend the number of candidates for lysosomal transporters, I took part to the validation step of an extensive proteomic study of the lysosomal membrane, which revealed forty-six new candidates for lysosomal transporters. I studied in more details TMEM104, SPINSTER, MFSD1, SLC37A2, TTYH3 and SNAT7. Proteins were overexpressed in HeLa cells to check for lysosomal localization. Then, their putative sorting motifs were mutated to misroute their expression to plasma membrane and to enable their functional study. No function could elucidate for the first five candidates.SNAT7 could not be misrouted to plasma membrane either, but, since it belonged to a family of transporters for glutamine, its function was studied by an indirect assay based on a lysosomal overload in amino acids and a direct transport measure on lysosome-enriched cellular fractions. Thus, SNAT7 was shown to be a lysosomal transporter selective for glutamine and asparagine.The function of SNAT7 is the nutrition of cancer cells was then studied. Many cancer cells use glutamine as their main source of carbon, nitrogen and energy. Because of insufficient blood supply, they use macropinocytosis to uptake extracellular proteins, which degradation in lysosomes generates glutamine. Then, glutamine is recycled to the cytosol. SNAT7 was shown to be critical in this process: in glutamine-dependent cancer cells, when SNAT7 expression is reduced, cells cannot obtain glutamine from extracellular proteins. Thus, blocking SNAT7 is a promising approach to target specifically the metabolism of cancer cells.

Schizophrenia is primarily treated with the use of antipsychotic medications. However, antipsychotics used have severe, dose-dependent side effects in schizophrenia patients. Therefore, there is a need for new adjunctive drugs that lower the effective dose of first line schizophrenia drugs and improve patient symptoms. Neuroinflammation is associated with microglial activation in schizophrenia, and increased tumor necrosis factor-alpha (TNF) has shown to be associated with Metabolic Syndrome in schizophrenia patients. A newly developed anti-neuroinflammatory, PD2024, reduces TNF-alpha action in vitro and in vivo, and has been shown to be well-tolerated in rat and dog studies with no adverse effects. The purpose of this research is to evaluate the effect of PD2024 in two well-defined schizophrenia models in rats. The neonatal quinpirole model has been established through administration of the dopamine D2-like agonist quinpirole (NQ) or saline control (NS) postnatally from days 1-21. NQ treatment results in increases of dopamine D2 receptor sensitivity throughout the animal’s lifetime without changing receptor number, mimicking a hallmark of schizophrenia. The polyinosinic:polycytidylic acid (Poly I:C) model is based on mimicking an increase immune response during early brain development, which has been shown to increase the prevalence of schizophrenia. Poly I:C (2 mg/kg) was administered during the neonatal period at postnatal days (P)5-7 to produce this effect. Both models were given PD2024 at 10mg/kg orally through the diet from P30-67. Prepulse inhibition (PPI) was used to test sensorimotor gating deficits in the rats. PPI has past research showing its use as a quantitative phenotype for evaluating schizophrenia-associated behavioral and neurobiological deficits. In our PPI test, rats are exposed to three different, randomly ordered noise trials. The trials included a pulse trial with a 120-decibel startle pulse, a prepulse trial with an auditory click at 73, 76, or 82-decibels, and a no stimulus trial without any additional noise. The rats were given 25 randomized trials, comprised of 5 pulse, 15 prepulse (5 each of 73, 76, and 82dB) and 5 no stimulus trials. Background noise was 70dB, and the rats were tested during adolescence (days 45-46) and adulthood (60-65). In NQ adolescent rats, PPI was significantly improved in the PD2024-treated compared to NQ controls. NQ-PD2024 and NS rats were statistically equivalent throughout the trials. These results were reflected in the NQ adult model as well. The Poly I:C adolescents treated with PD2024 also demonstrated improved PPI performance compared to Poly I:C controls. This improvement was also shown in the adult Poly I:C rats. Overall, the PPI deficits in both models improved between 15 to 30% in adolescence and adulthood. These results indicate that PD2024 is effective in treating schizophrenia-associated behaviors.

Over 10 million people are affected by major depressive disorder (MDD) in the U.S. annually. Unfortunately, about 1/3 of these people do not achieve adequate remission of symptoms with current antidepressant drugs. It is expected that an improved understanding of the pathobiology of depression will result in the development of more effective antidepressant treatments. Research by this lab in recent years has provided evidence of elevated DNA damage in brain white matter in MDD, discovered by studying brain tissues from human brain donors that had an active diagnosis of MDD at the time of death and age-matched control donors who had no psychiatric illness. Accompanying this DNA damage was an elevation of gene expression of DNA base excision repair enzymes in white matter oligodendrocytes, a major cell type in brain white matter. In addition, gene expression of antioxidant genes in these oligodendrocytes was significantly lower in MDD than in control donors, suggesting that these cells were especially susceptible to the damaging effects of oxidative stress in MDD. This initial data was generated by measuring gene expressions in oligodendrocytes captured from two specific regions of white matter in the brain, the frontal cortex, and amygdala. In the present study, we designed experiments to determine whether these effects are found in oligodendrocytes in other areas of the brain in MDD and to determine whether another cell type in the brain, neurons, are similarly affected. Towards these aims, oligodendrocytes from two other brain regions (occipital cortical white matter and brainstem locus coeruleus) were captured by laser microdissection from MDD and control donors. In addition, CA1 pyramidal neurons were captured from the anterior hippocampus of MDD and control donors. We chose to specifically study hippocampal CA1 pyramidal neurons because these neurons are normally sensitive to oxidative stress, and reasoned that these cells would be among brain neurons most likely affected by conditions of elevated oxidative stress in MDD. Approximately 500 cells were captured from each brain area using immunohistochemically-guided laser capture microdissection. RNA isolated from these cells was converted to cDNA by reverse transcription and subjected to quantitative polymerase chain reactions (PCR). Statistically significant reductions in antioxidant gene expression was observed in oligodendrocytes from MDD donors as compared to control donors regardless of the brain area from which the cells were captured. In contrast, no significant changes in antioxidant gene expression were observed in CA1 pyramidal neurons from MDD donors. Additionally in contrast to findings in oligodendrocytes, levels of gene expression of the DNA repair enzyme, poly(ADP-ribose) polymerase 1 (PARP1) in hippocampal CA1 pyramidal neurons from MDD donors was similar to that from control donors. These findings demonstrate that pathological DNA damage and repair mechanisms occur in brain oligodendrocytes throughout the brain, and similar mechanisms do not appear to affect hippocampal neurons. A better understanding of the cellular systems engaged by oxidative damage to oligodendrocytes in MDD has the potential to lead to the identification of unique targets for the development of novel antidepressant drugs.

Neuronal networks, derived from mouse embryonic frontal cortex (FC) tissue grown on microelectrode arrays, were used to investigate effects of gentamicin pretreatment on pharmacological response to the L-type calcium channel blocker, verapamil. Gentamicin is a broad spectrum antibiotic used to control bacterial contamination in cell culture. The addition of gentamicin directly to medium affects the pharmacological and morphological properties of the cells in culture. A reproducible dose response curve to verapamil from untreated cultures was established and the mean EC50 was calculated to be 1.5 ± 0.5 μM (n=10). 40 μM bicuculline was added to some cell cultures to stabilize activity and verapamil dose response curves were performed in presence of bicuculline, EC50 1.4 ± 0.1 μM (n=9). Statistical analysis showed no significant difference in verapamil EC50s values obtained in presence of bicuculline and hence the data was combined and a standard verapamil EC50 was calculated as 1.4 ± 0.13 μM (n=19). This EC50 was then used to compare verapamil EC50s obtained from neuronal cell cultures with chronic and acute exposures to gentamicin. FC cultures (21- 38 days old) were found to be stable in presence of 2300 μM gentamicin. The recommended concentration of gentamicin for contamination control is 5uL /1 ml medium (108 μM). At this concentration, the verapamil EC50 shifted from 1.4 ± 0.13 μM to 0.9 ± 0.2 μM. Given the limited data points and only two complete CRCs, statistical comparison was not feasible. However, there is a definite trend that shows sensitization of cells to verapamil in presence of gentamicin. The cultures exposed to 108 μM gentamicin for 5 days after seeding showed loss of adhesion and no data could be collected for pharmacological analysis. To conclude, acute gentamicin exposure of neuronal cell cultures causes increased sensitivity to verapamil and chronic or long term exposure to gentamicin may cause loss of adhesion of the cell culture by affecting the glial growth. The effect of chronic exposure to gentamicin on pharmacological responses to verapamil remains inconclusive.