Cellular electroporation induces dedifferentiation in intact newt limbs

AbstractNewts have the remarkable ability to regenerate lost appendages including their forelimbs, hindlimbs, and tails. Following amputation of an appendage, the wound is rapidly closed by the migration of epithelial cells from the proximal epidermis. Internal cells just proximal to the amputation plane begin to dedifferentiate to form a pool of proliferating progenitor cells known as the regeneration blastema. We show that dedifferentiation of internal appendage cells can be initiated in the absence of amputation by applying an electric field sufficient to induce cellular electroporation, but not necrosis or apoptosis. The time course for dedifferentiation following electroporation is similar to that observed following amputation with evidence of dedifferentiation beginning at about 5 days postelectroporation and continuing for 2 to 3 weeks. Microarray analyses, real-time RT-PCR, and in situ hybridization show that changes in early gene expression are similar following amputation or electroporation. We conclude that the application of an electric field sufficient to induce transient electroporation of cell membranes induces a dedifferentiation response that is virtually indistinguishable from the response that occurs following amputation of newt appendages. This discovery allows dedifferentiation to be studied in the absence of wound healing and may aid in identifying genes required for cellular plasticity

PhD

dissertationDrug addiction is a serious problem for modern societies worldwide. There are remarkable costs associated with this problem, from the direct costs of the substance abuse treatment and prevention healthcare infrastructure to the less direct implications for the criminal justice system and social welfare programs. For individuals there is a loss of health, livelihood, interpersonal relationships, freedom, and ultimately life. The hallmark behavioral feature of addiction is a pattern of intermittent relapse lasting for decades after initial substance abuse. The research community has made a substantial investment of time and resources to better understand the etiology and fundamental features underlying addiction and relapse. Modern theories posit that addiction involves a recurring compulsion by an individual to engage in drug-seeking and drug-taking behaviors despite significant negative consequences. In humans, exposure to drug-associated stimuli and environmental contexts is known to induce an intense state of craving, independent of the length of time since acute drug exposure. Furthermore, neuroimaging studies of humans have suggested that basal ganglia structures may be involved in craving. Progress in the study of basal ganglia-mediated learning and memory has begun to outline a neuroanatomical substrate for the transition of egocentric, selfinitiated goal-directed behavior, to allocentric or externally driven behavior arising from exposure to environmental contexts or particular stimuli. While many elegant experiments have addressed the ability of drug-associated environmental contexts to affect behavior, the ability of discrete drug-associated sensory cues to affect behavior has been less thoroughly addressed. Discrete cues can become associated with drug use by two major operant learning mechanisms that vary in their temporal relationship to the subjective perception of primary reinforcement. The experiments described in this dissertation were designed to identify the extent to which discriminative stimuli and conditioned reinforcers activate the same or different populations of neurons in the dorsal striatum, the main input nucleus of the mammalian basal ganglia. This work has examined, in a novel experimental model of relapse, the capability of discrete sensory cues to elicit reinstatement of drug-seeking behavior and the expression of learning and memory related genes associated with this behavior

Lack of increased immediate early gene expression in rats reinstating cocaine-seeking behavior to discrete sensory cues.

Drug-seeking behavior elicited by drug-associated cues contributes to relapse in addiction; however, whether relapse elicited by drug-associated conditioned reinforcers (CR) versus discriminative stimuli (DS) involves distinct or overlapping neuronal populations is unknown. To address this question, we developed a novel cocaine self-administration and cue-induced reinstatement paradigm that exposed the same rats to distinct cocaine-associated CR and DS. Rats were trained to self-administer cocaine in separate sessions. In one, a DS signaled cocaine availability; in the other, cocaine delivery was paired with a different CR. After extinction training and reinstatement testing, where both cues were presented in separate sessions, rats were sacrificed and processed for cellular analysis of temporal activity by fluorescent in situ hybridization (CatFISH) for activity regulated cytoskeleton-associated protein (Arc) mRNA and for radioactive in situ hybridization for Arc and zif268 mRNAs. CatFISH did not reveal significant changes in Arc mRNA expression. Similar results were obtained with radioactive in situ hybridization. We have shown that while rats reinstate drug seeking in response to temporally discrete presentations of distinct drug-associated cues, such reinstatement is not associated with increased transcriptional activation of Arc or zif268 mRNAs, suggesting that expression of these genes may not be necessary for cue-induced reinstatement of drug-seeking behavior

Representative images from radioactive <i>in situ</i> hybridization film autoradiograms of <i>Arc-³⁵S</i> mRNA expression.

<p>Images of film autoradiograms from a single cohort of animals showing <i>Arc-³⁵S</i> mRNA expression in black for caged control (<b>CC</b>), experimental (<b>EXP</b>), drug control (<b>DC</b>), stimulus control (<b>SC</b>), and novelty control (<b>NC</b>) animals in various subregions of the (<b>A</b>) prefrontal cortex and (<b>B</b>) striatum.</p

Densitometric analysis of <i>Arc</i> mRNA expression.

<p><i>Arc</i> mRNA expression (integrated density) determined from analysis of film autoradiograms. Data are means ± SEM (n = 4–6/group). <b>*</b> Significantly different from CC, p<0.05.</p

Percentage of <i>Arc</i> mRNA-positive neurons showing dual nuclear and cytoplasmic expression (“dual-associated”).

<p>Percentage of <i>Arc mRNA</i>-positive neurons with dual (<i>i.e</i>. both nuclear and cytoplasmic) <i>Arc</i> mRNA expression by region analyzed in caged control (<b>CC</b>), experimental (<b>EXP</b>), novelty control (<b>NC</b>), drug control (<b>DC</b>), and stimulus control (<b>SC</b>) rats (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072883#s2" target="_blank">Methods</a> for full description of different groups). Data are mean percentage ± SEM (n = 4–6) of <i>Arc</i> mRNA-positive neurons showing dual-associated expression. <b>*</b> Significantly different from all other groups, p<0.05.</p

Catfish results.

<p>Catfish results.</p

Cocaine self-administration and extinction behavior.

<p>Experimental rats earned high and stable levels of self-administered cocaine infusions (0.25 mg cocaine-HCL/50 µL 0.9% saline/2-sec) in both DS- and CR-associated session segments and required approximately 25 sessions for daily infusion rates to stabilize. (<b>A</b>) The 15 sessions preceding and culminating in attainment of criterion (3 consecutive sessions with <15 coefficient of variability) are shown. In addition to demonstrating high levels of active-lever pressing during self-administration (<b>B</b>; <b>SA-15</b>), EXP rats also extinguished active-lever pressing by the last of 14 extinction sessions (<b>B</b>; <b>EXT-14</b>). Data are means ± SEM for (<b>A</b>) numbers of cocaine infusions per two-hour segment and (<b>B</b>) lever presses per 4.5-hour session.</p

Cocaine self-administration training, cue-induced reinstatement, and catFISH expression profiles.

<p>Each session began with a 5-min delay and lasted for four hours and 35 min. EXP rats had a two-hr DS or CR session followed by a 30-min time out and the alternate session. (<b>A</b>) <i>Discriminative stimulus training</i> The DS (house light) was illuminated for up to two min. Contingent on FR1 active-lever pressing, the DS extinguished and the rat received a cocaine infusion followed by a variable-interval time out (VI; mean  = 8 min). (<b>B</b>) <i>Conditioned reinforcer training</i> EXP rats received a cocaine infusion paired with a complex CR (illumination of tricolor LEDs and tone) contingent on FR5 active-lever pressing. After cocaine infusion and CR exposure there was a 20-sec non-signaled time out. (<b>C</b>) <i>Cue-induced reinstatement</i> After extinction, rats remained in the operant chambers overnight. The next morning, contingent on an EXP active-lever press, the CR was presented for 5 sec. For the next 5 min, the CR was presented for two sec, contingent on FR1 active-lever pressing. Next, there was a 20-min time out. Then the DS was illuminated for two min, extinguished for one min, re-illuminated for two min, and finally extinguished. (<b>C</b>) <i>CatFISH analysis examples</i> Neurons showing one or two intranuclear foci of <i>Arc</i> mRNA expression (white arrows; “<b>FOCI</b>”), cytoplasmic <i>Arc</i> mRNA expression (white asterisks; “<b>CYTO</b>”), or both intranuclear and cytoplasmic <i>Arc</i> mRNA expression (“<b>DUAL</b>”). Neurons expressing only foci of <i>Arc</i> mRNA (i.e. those activated 5 min prior to sacrifice) are referred to as “DS-associated,” neurons expressing only cytoplasmic <i>Arc</i> mRNA (i.e. those activated 30 min prior to sacrifice) are referred to as “CR-associated,” and neurons expressing both intranuclear foci and cytoplasmic <i>Arc</i> mRNA expression (i.e. those activated both 5 and 30 min prior to sacrifice) are referred to as “Dual associated.”</p

Densitometric analysis of <i>zif268</i> mRNA expression.

<p><i>zif268</i> mRNA expression (integrated density) determined from analysis of film autoradiograms. Data are means ± SEM (n = 4–6/group). <b>*</b> Significantly different from CC, p<0.05.</p