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Federal government websites often end in. The site is secure. Preview improvements coming to the PMC website in October Learn More or Try it out now. Cocaine self-administration SA in rats dysregulates glutamatergic signaling in the prelimbic PrL cortex and glutamate release in the nucleus accumbens NA core, promoting cocaine seeking. PrL adaptations that affect relapse to drug seeking emerge during the first week of abstinence; switching from an early 2 hr hypoglutamatergic state to a later 7 days hyperglutamatergic state. Different interventions that normalize glutamatergic signaling in PrL cortex at each timepoint are necessary to suppress relapse. We hypothesized that plasticity-related proteins that regulate glutamatergic neurotransmission as well as dendritic spine morphology would be biphasically regulated during these two phases of abstinence in PrL cortical neurons projecting to the NA core PrL-NA core. A combinatorial viral approach was used to selectively label PrL-NA core neurons with an mCherry fluorescent reporter. Male rats underwent two weeks of cocaine SA or received yoked-saline infusions and were perfused either 2 hr or 7 days after the final SA session. Our findings reveal biphasic, abstinence duration-dependent alterations in structural plasticity and relapse-related proteins in the PrL-NA core pathway after cocaine SA. Cocaine self-administration SA leads to a myriad of adaptations in plasticity-related proteins PRPs that mediate glutamatergic transmission in the prelimbic PrL cortex. These neuroadaptations change dramatically during the first week of abstinence. Two hours after cocaine exposure ends i. In contrast, after one week of abstinence i. These findings indicate that biphasic alterations in glutamatergic transmission in the PrL cortex occur during early withdrawal and abstinence, requiring different time-dependent interventions to decrease drug seeking. However, it is unknown whether the global changes in PrL PRPs are expressed by pathway-specific projections to subcortical targets or if the changes in the expression of synaptic PRPs AMPA receptors are associated with morphometric alterations in dendritic spines that have previously been linked to relapse propensity Gipson et al. Changes in dendritic spine morphology and AMPA receptor expression in spine heads are associated with alterations in glutamatergic transmission. Further, GluA1-containing AMPA receptors are preferentially incorporated into mushroom-type dendritic spines that stabilize synapses following learning Matsuo et al. Thus, cocaine-induced adaptations in glutamate transmission are likely associated with altered dendritic spine morphology and AMPA receptor expression in spine heads. Although cocaine-induced adaptations in structural plasticity within the NA core are well-documented Gipson et al. Golgi staining studies have demonstrated elevated apical spine density on layer V PrL neurons after one month of abstinence Robinson et al. Recently, dye filling techniques and higher resolution imaging studies have shown that after two weeks of abstinence from cocaine SA, global PrL apical spine density i. Collectively, these data demonstrate that changes in structural plasticity in the PrL cortex after cocaine SA depend on the type of pyramidal neurons analyzed, the duration of abstinence and, perhaps most importantly, the technological strategy used to measure dendritic spine morphology. However, it is now accepted that PrL neurons projecting to distinct subcortical targets display hetereogeneity regarding the encoding of reward-associated stimuli Otis et al. Accordingly, to achieve cell-type and projection specificity, viral vectors and Cre-dependent fluorescent reporters have recently been used to label neurons for spine analyses in the nucleus accumbens Dos Santos et al. Recently, we found that a combinatorial viral approach fully labels pyramidal neurons in the PrL cortex with pathway specificity that can be combined with immunohistochemical detection of AMPA receptors in different dendritic subcompartments, allowing for analysis of dendritic spine morphology and associated protein expression in PrL cortical neurons projecting to the NA core PrL-NA core. In the current study, we focused our attention specifically on the distal apical tuft of layer V PrL-NA core neurons because chronic stress produces a specific reduction in distal apical tuft spine density in layer V PrL cortical neurons Liu and Aghajanian and recent evidence indicates a relationship between abstinence from cocaine SA, stress, and decreased PrL cortical apical dendrite spine density Radley et al. Thus, structural modifications in the distal tuft may directly impact PrL-NA core neuronal physiology. Given that PrL glutamatergic transmission switches from a putatively hypoactive to a hyperactive state as a function of the duration of abstinence from cocaine SA Dennis et al. All rats weighed —g at the time of surgery. Upon arrival, rats were allowed at least 3 days of acclimation to the vivarium. During this time, they were provided standard rat chow Harlan; Indianapolis, IN and water ad libitum. On the day of surgery, rats were injected i. Following catheterization, rats were secured in a stereotaxic apparatus Kopf Instruments, Tujunga, CA for intra-cranial virus microinjections. All viral procedures and constructs used in this study were approved by the Medical University of South Carolina Institutional Biosafety Committee. All rats received a single microinjection 0. Injections were performed over a period of 5 minutes 0. Following surgery, bore holes were sealed with dental acrylic, and the incision was sutured closed. Rats were allowed at least 5 days of recovery, during which food and water were available ad libitum prior to beginning behavioral training. Self-administration was conducted in standard Med Associates operant chambers Fairfax, VT containing two retractable levers. Inactive lever presses had no programmed consequence but were recorded. Yoked saline controls received a single non-contingent bolus of 0. In Experiment 2, rats were perfused one week after the final cocaine SA session following forced homecage abstinence where food and water were provided ad libitum , and brains were post-fixed for 24 hours. To match virus expression duration between experiments, rats in experiment 1 were given an extra week of recovery after surgery prior to beginning SA. Immunohistochemistry was performed according to previously published protocols Scofield et al. Briefly, sections were blocked with 0. Z-stacks were then imported to Imaris. The spot tool in Imaris was used, and detection threshold and spot size were empirically determined and maintained constant for each measurement. Overall imaging parameters including laser power, gain, pinhole, and optical section thickness were set empirically prior to imaging and were held constant for the remainder of the experiment. Emitted photons were collected using high-sensitivity hybrid detectors HyD. Images were acquired using a 63X oil immersion objective 1. Images were acquired using sub airy unit pinhole sizes. These parameters were selected to obtain optimal deconvolution as recommended by Huygens essential deconvolution software Hilversum, The Netherlands. The resulting set of parameters allows for a minimum resolution of nm XY by nm Z in our reconstructions. One dendritic spine segment was imaged per neuron each with an accompanying GluA1 or GluA2 channel collected. Z-stacks were cropped in 3D to isolate individual dendrites, and a 3D space-filling model was generated to isolate voxels corresponding to the mCherry-positive dendrite of interest. The filament module was used to semi-manually trace identified dendritic spines as described previously Trantham-Davidson et al. Dendritic spine head diameter was calculated using an automated threshold set by Imaris. Filament analysis uses the predefined boundaries of the spine heads relative to the dendritic shaft to isolate specific channels of interest in dendritic subcompartments. For colocalization analyses, a region of interest ROI containing only the mCherry dendrite was used. The intensity value at which a voxel could be considered coregistered was set empirically by a blind investigator for both channels as described previously Scofield et al. We recorded the percent of signal above threshold to determine whether differences in the intensity of signal impacted coregistry analyses for GluA1, GluA2, and mCherry. Electron microscopy studies indicate that a significant amount of GluA2 expression is present in both dendritic shafts and spine heads Vissavajjhala et al. To determine specific spine head bins that may account for overall alterations in spine head diameter, the number of spines within each bin was normalized to the total number of spines and compared between groups. In a minority of instances when sections from individual animals showed mCherry neurons labeled in one for GluA1 but not GluA2, and vice versa, the spine morphometry data for that animal consisted of the run in which mCherry neurons were labeled, but the colocalization data for that animal for that specific protein probed was not included. All analyses were performed by an individual blind to experimental groups. Cumulative frequency distribution data were analyzed to determine general shifts in spine head diameter for each group using a Kolmogorov-Smirnov non-parametric test. Sample sizes for each group for each experiment are stated in the respective figure legends. The design and timeline for Experiments 1 and 2 are shown in Fig. In Experiment 1, 16 rats underwent cocaine SA or received yoked-saline infusions and were perfused 2 hr after the final session. One yoked-saline rat was excluded due to a missed NA core placement. Two cocaine animals were excluded due to failed catheters. In Experiment 2, 16 rats underwent 12—14 days of cocaine SA or received yoked-saline infusions. One yoked-saline rat did not finish the experiment due to stress reactions when handled and another was removed from analyses due to insufficient virus expression. One cocaine SA rat was removed as a statistical outlier in colocalization analyses. Experimental timeline, virus expression, and cocaine SA data. Solid lines and closed symbols indicate active lever presses and dotted lines with open symbols indicate inactive lever presses. Dotted lines indicate lateral ventricle top , anterior commissure ac, center and border of NA core and NA shell. Inset corresponds to a representative dendritic spine segment taken from the distal apical tuft. Bottom right — GluA2-mCherry coregistered voxels in dendritic spine heads yellow and dendritic shaft white. SA: self-administration. In Experiment 1, rats underwent cocaine SA or received yoked-saline infusions and were perfused two hours after the final session. Representative images are shown in Fig. S2a-b , respectively, indicating that differences in signal intensity are unlikely to be the cause of the observed effect of cocaine. To address this hypothesis, we analyzed 52 dendritic segments 1, total spines from 7 yoked saline rats and 54 dendritic segments 2, total spines from 6 cocaine SA rats. From these 13 rats, we sampled an average of 8. Importantly, we did not observe significant differences between groups in the average spine terminal point intensity Fig. S3a , indicating that differences in mCherry signal intensity are unlikely to contribute to the effects observed on spine d H. Specifically, cocaine SA rats displayed a significant increase in the percentage of total spines with head diameters between 0. Representative dendritic images are shown in Fig. One yoked-saline animal showed no mCherry-labeled neurons and was not included in the analysis. Representative dendritic segments are shown in Fig. Further, we also did not observe significant differences in average GluA1-IR intensity within mCherry dendrites as shown in Fig. As an extension of these analyses, sections from all animals were immunostained for mCherry and GluA2. One yoked-saline animal was excluded from analysis due to immunohistochemical detection issues. S2c-d respectively. To address this hypothesis, we analyzed 44 dendritic segments 1, total spines from 6 yoked saline rats and 48 dendritic segments 1, total spines from 7 cocaine SA rats. From these 13 animals, we sampled an average of 7. Cocaine SA rats showed a reduction in the percentage of total spines with d H between 0. Representative dendritic segments are shown in Figure 4h. There was no difference in the average spine terminal point intensity between groups as shown in Fig. Sections from one cocaine animal did not show virus expression in the run processed for GluA1. This study shows that cocaine SA produces an abstinence duration-dependent, biphasic alteration in nuclear and dendritic spine PRPs and dendritic spine morphology of PrL-NA core neurons. However, both AMPA receptor subunits were reduced in putative mushroom-type spines. Figure 6 summarizes these adaptations at both abstinence timepoints. Diagram showing abstinence duration-dependent biphasic alterations in spine morphology and plasticity-related proteins in PrL-NA core neurons. This chain of events likely leads to dysfunctional encoding of cocaine-predictive stimuli, ultimately driving relapse following reintroduction of cocaine-paired contexts. This evidence extends previous data from our laboratory showing that cocaine SA leads to a decrease in phospho-protein signaling in the PrL cortex during early withdrawal Sun et al. Alterations in Fos and CREB expression are linked to changes in dendritic spine morphological properties Middei et al. Albeit correlative, we found that animals which had lower spine d H i. This finding is consistent with findings showing that expression of a dominant negative phospho-deficient mutation of pCREB S prevents dendritic spine formation associated with contextual fear learning Middei et al. Moreover, this mutation decreases basal and learning-induced GluA1 synaptic incorporation that is associated with a decrease in long-term potentiation and an increase in long-term depression in the hippocampus Middei et al. There are precedents for differential regulation of AMPA receptor expression; these two subunits undergo differential trafficking in dendrites and spines due to heterogenous interactions with subunit-specific AMPA receptor-interacting proteins Shepherd and Huganir ; Anggono and Huganir Although our findings indicate that early withdrawal differentially regulates total GluA1-IR and GluA2-IR in PrL-NA core neurons, our data demonstrating that both subunits were reduced in enlarged dendritic spines, in which the majority of AMPA receptor expression and function occurs Matsuzaki et al. Future studies will examine the molecular mechanisms underlying the cocaine-induced dendritic spine morphological adaptations and associated GluA1 and GluA2 mobilization. One interpretation of these data is that augmentation of PRPs and spine d H render PrL-NA core neurons hyper-excitable to glutamate inputs during early abstinence, increasing the likelihood that these neurons are selectively involved in the memory trace encoding the salience of the cocaine-conditioned context. This interpretation is supported by findings showing that overexpression of CREB in a subset of amygdala neurons increases the likelihood that these neurons are involved in a neuronal ensemble encoding a fear memory context Han et al. The increase in nuclear pCREB and the potential for subsequent increased excitability following abstinence from cocaine SA are supported by recent data indicating that extinction from cocaine SA increases the intrinsic excitability of layer V PrL neurons, an effect required for cue-induced reinstatement Sepulveda-Orengo et al. As discussed above, stabilization of individual excitatory synapses may be one function of enhanced pCREB in PrL-NA core neurons, which is associated with changes in morphological properties of dendritic spines and associated AMPA receptor expression Kasai et al. In contrast to early withdrawal, cocaine SA followed by early 1 week or more prolonged two weeks to one month abstinence has previously been shown to alter structural plasticity within the PrL cortex Radley et al. However, conflicting reports have emerged regarding the nature and extent of abstinence-induced PrL structural plasticity. Our data indicate that cocaine SA followed by one week of abstinence decreased the density of PrL-NA core neuron apical dendritic spines, yet increased the spine d H. The extant literature on layer V PrL cortical structural plasticity at this timepoint reports increased layer V basal thin spine density, but decreased synapse number and overall dendritic complexity Rasakham et al. Our results are complementary to this finding because our data derive from pathway-specific structural modifications. Moreover, the region of dendrite investigated is also an important factor, as inputs to the apical tuft Liu and Aghajanian and basal dendrites Liu et al. Finally, our findings are in general agreement with recent experiments utilizing similar methodology demonstrating decreased apical spine density of PrL neurons specifically projecting to D1-expressing MSNs in the NA core following two weeks of abstinence from a sensitizing regimen of cocaine administration Barrientos et al. Regarding AMPA receptor plasticity during early abstinence, in contrast to early withdrawal, we found decreased dendritic shaft-specific, but elevated spine head-specific, GluA1-IR and increased spine head-specific GluA2-IR. These findings demonstrate dendritic compartment-specific changes in GluA1-IR in PrL-NA core neurons after one week of abstinence that are distinct from those that occur during early withdrawal. Although it has been well-characterized that GluA2-lacking AMPA receptors in the NA core play a central role in incubation of cocaine seeking after prolonged abstinence in long-access SA paradigms Conrad et al. Regardless, increased AMPA receptor expression in enlarged dendritic spines likely disposes these dendritic compartments to be hypersensitive to glutamate release given that spines with enlarged volume show greater AMPA receptor-mediated currents Matsuzaki et al. Moreover, because we found an overall decrease in spine density, but increased spine d H associated with increased AMPA-IR in enlarged spines, it is intriguing to hypothesize that specific inputs to the distal apical tuft of PrL-NA core neurons may be potentiated whereas others are depressed. This is supported by previous work showing that spines adjacent to active synapses have an accumulation of AMPA receptors leading to a lack of lateral diffusion i. Layer V prefrontal cortical pyramidal neurons receive dense glutamatergic inputs arising from cortico-cortical and thalamocortical neurons Berendse and Groenewegen The axon boutons of thalamocortical neurons are under the control of several neuromodulators, including orexin, acetylcholine, and serotonin. Finally, serotonin application to apical, but not basal, layer V PrL cortical dendrites elicits EPSCs in layer V PrL cortical neurons Aghajanian and Marek , an effect eliminated by chronic stress, which leads to apical dendrite atrophy Liu and Aghajanian Given the link between chronic stress-induced dendritic atrophy and cocaine SA-induced spine morphometric adaptations in the PrL cortex Radley et al. One possible scenario is that daily cocaine SA sessions lead to time-dependent increases in glutamate release onto layer V PrL-NA core apical dendritic spines arising from thalamocortical neurons, promoting encoding of cocaine-associated sensory stimuli. However, PrL-NA core neurons may compensate for augmented glutamate release by reducing the size of dendritic spines and AMPA receptor occupancy in apical spine heads, leading to reduced phospho-protein signaling, during early withdrawal from cocaine SA. The identity and functional role of the salient inputs that are responsible for the observed effects of cocaine SA at both abstinence timepoints will be a focus of future investigation. The data reported herein are consistent with the global changes in PrL cortex we have previously reported and suggest that the changes in PRPs and dendritic spines in the PrL-NA core pathway during early withdrawal also contribute to relapse and would be reversed by intra-PrL BDNF. Glutamate uncaging at single spines of CA1 pyramidal neurons rapidly increases dendritic spine head size, which is dependent on BDNF-TrkB signaling, and exogenous BDNF application combined with glutamate uncaging produces a more sustained increase in spine head size relative to glutamate uncaging alone Tanaka et al. Thus, it is likely that a single BDNF microinfusion increases AMPA receptor accumulation in dendritic spines which likely requires sustained spine head-enlargement. However, other relevant downstream pathways related to the regulation of actin polymerization by BDNF-TrkB signaling are likely also involved. Ultimately, these effects may lead to a normalization of glutamate transmission in the PrL-NA core pathway. This hypothesis is supported by previous data indicating that a single BDNF microinfusion in the PrL cortex immediately after the final SA session leads to a long-term normalization of extracellular glutamate in the NA core and prevents cocaine prime-induced elevations in extracellular glutamate Berglind et al. In contrast, over the course of one week of abstinence, PrL-NA core neurons appear to undergo allostatic alterations in structure and function in order to counteract the early withdrawal effects of cocaine on PRPs, leading to enhanced encoding of cocaine-conditioned stimuli that takes time to develop. Instead, adaptations in PrL-NA core structural plasticity and PRPs after one week of abstinence would likely be responsive to PKA inhibition as our previous data derived from western blotting show Sun et al. In future studies, we will examine additional timepoints during abstinence and the effects of BDNF and PKA inhibition on associated structural and synaptic plasticity. The functional relevance of these changes in PrL-NA core neurons to drug seeking is also the subject of further investigation. Our findings link previously described biphasic alterations in glutamatergic signaling occurring globally in the PrL cortex during early withdrawal and early abstinence with similar adaptations in the PrL-NA core pathway. These adaptations occur not only in the nucleus of PrL-NA core neurons, but also at the level of individual dendritic spines, leading to differential compartmentalization of AMPA receptors in spines and dendritic shafts at different times after cocaine exposure. Given that there is remarkable dichotomy in PrL-NA core adaptations at the two timepoints studied, we hypothesize that the altered structure and, presumably diminished function, of PrL-NA core neurons during early withdrawal likely trigger the subsequent enlargement of dendritic spines, induction of pCREB, and accumulation of AMPA receptors in enlarged spines in these neurons after one week of abstinence Fig. Thus, the abstinence duration-dependent adaptations in PrL-NA core neurons provide a novel way of interpreting PrL-NA core dysfunction pertaining to relapse vulnerability. Moreover, these findings suggest that treatments for relapse vulnerability in clinical populations should consider the timing at which interventions occur given the heterogeneity in PRP adaptions in critical brain regions over the course of abstinence. We thank Jordan Hopkins and Kaylee Hooker for excellent technical assistance. All procedures performed in studies involving animals were in accordance with the ethical standards of the institution or practice at which the studies were conducted. This article does not contain any studies with human participants performed by any of the authors. The authors declare that they have no conflict of interest. As a library, NLM provides access to scientific literature. Brain Struct Funct. Author manuscript; available in PMC Mar Address correspondence to: Dr. Jacqueline F. PMC Copyright notice. The publisher's final edited version of this article is available at Brain Struct Funct. Associated Data Supplementary Materials Suppl. Abstract Cocaine self-administration SA in rats dysregulates glutamatergic signaling in the prelimbic PrL cortex and glutamate release in the nucleus accumbens NA core, promoting cocaine seeking. Keywords: cocaine, prelimbic cortex, nucleus accumbens, dendritic spines, AMPA receptors, glutamate. Introduction Cocaine self-administration SA leads to a myriad of adaptations in plasticity-related proteins PRPs that mediate glutamatergic transmission in the prelimbic PrL cortex. Viral constructs and surgery On the day of surgery, rats were injected i. Dendritic Spine Imaging. Imaris 3D reconstruction, morphological analyses, and colocalization Following deconvolution, Z-stacks were imported into BitPlane Imaris Zurich, Switzerland, version 9. Open in a separate window. Fig 1. Fig 2. Fig 3. Fig 4. Fig 5. Discussion This study shows that cocaine SA produces an abstinence duration-dependent, biphasic alteration in nuclear and dendritic spine PRPs and dendritic spine morphology of PrL-NA core neurons. Fig 6. Potential inputs to PrL-NA core neurons and functional implications Layer V prefrontal cortical pyramidal neurons receive dense glutamatergic inputs arising from cortico-cortical and thalamocortical neurons Berendse and Groenewegen Conclusions Our findings link previously described biphasic alterations in glutamatergic signaling occurring globally in the PrL cortex during early withdrawal and early abstinence with similar adaptations in the PrL-NA core pathway. Supplementary Material Suppl Click here to view. Footnotes Compliance with ethical standards All procedures performed in studies involving animals were in accordance with the ethical standards of the institution or practice at which the studies were conducted. References Aghajanian GK, Marek GJ Serotonin induces excitatory postsynaptic potentials in apical dendrites of neocortical pyramidal cells. Curr Opin Neurobiol 22 — Neuroscience — Biological Psychiatry. 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