Impact of Perineuronal Net Removal in the Rat Medial Prefrontal Cortex on Parvalbumin Interneurons After Reinstatement of Cocaine Conditioned Place Preference.

Parvalbumin (PV)-positive cells are GABAergic fast-spiking interneurons that modulate the activity of pyramidal neurons in the medial prefrontal cortex (mPFC) and their output to brain areas associated with learning and memory. The majority of PV cells within the mPFC are surrounded by a specialized extracellular matrix structure called the perineuronal net (PNN). We have shown that removal of PNNs with the enzyme chondroitinase-ABC (Ch-ABC) in the mPFC prevents the consolidation and reconsolidation of cocaine-associated conditioned place preference (CPP) memories. Here we examined the extent to which retrieval of a CPP memory during cocaine-primed reinstatement altered the levels and function of PV neurons and their surrounding PNNs during the reconsolidation period. We further determined the extent to which PNN removal prior to reinstatement altered PV intensity levels and PV cell function. Male Sprague-Dawley rats were trained for cocaine-induced conditioned place preference (CPP) followed by extinction training, microinjection of Ch-ABC in the prelimbic PFC, and cocaine-induced reinstatement. Rats were sacrificed immediately prior to reinstatement or at 2 h, 6 h, or 48 h after reinstatement for immunohistochemistry or 2 h later for electrophysiology. Our findings indicate that PNN removal only partially diminished reinstatement. Cocaine-primed reinstatement produced only minor changes in PNN or PV intensity in vehicle controls. However, after PNN removal, the intensity of remaining PNN-surrounded PV cells was decreased at all times except at 2 h post-reinstatement, at which time cocaine increased PV intensity. Consistent with this, in vehicle controls, PV neurons naturally devoid of PNNs showed a similar pattern to Ch-ABC-treated rats prior to and after cocaine reinstatement, suggesting a protective effect of PNNs on cocaine-induced changes in PV intensity. Using whole-cell patch-clamp, cocaine-primed reinstatement in Ch-ABC-treated rats decreased the number of elicited action potentials but increased excitatory synaptic transmission, which may have been compensatory. These findings suggest that without PNNs, cocaine-induced reinstatement produces rapid changes in PV intensity and PV cell excitability, which may in turn regulate output of the mPFC post-memory retrieval and diminish the maintenance of cocaine memory during reconsolidation.

Diurnal changes in perineuronal nets and parvalbumin neurons in the rat medial prefrontal cortex.

Perineuronal nets (PNNs) surrounding fast-spiking, parvalbumin (PV) interneurons provide excitatory:inhibitory balance, which is impaired in several disorders associated with altered diurnal rhythms, yet few studies have examined diurnal rhythms of PNNs or PV cells. We measured the intensity and number of PV cells and PNNs labeled with Wisteria floribunda agglutinin (WFA) and also the oxidative stress marker 8-oxo-deoxyguanosine (8-oxo-dG) in rat prelimbic medial prefrontal cortex (mPFC) at Zeitgeber times (ZT) ZT0 (lights-on, inactive phase), ZT6 (mid-inactive phase), ZT12 (lights-off, active phase), and ZT18 (mid-active phase). Relative to ZT0, the intensities of PNN and PV labeling were increased in the dark (active) phase compared with the light (inactive) phase. The intensity of 8-oxo-dG was decreased from ZT0 at all times (ZT6,12,18). We also measured GAD 65/67 and vGLUT1 puncta apposed to PV cells with and without PNNs. There were more excitatory puncta on PV cells with PNNs at ZT18 vs. ZT6, but no changes in PV cells without PNNs and no changes in inhibitory puncta. Whole-cell slice recordings in fast-spiking (PV) cells with PNNs showed an increased ratio of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor:N-methyl-D-aspartate receptor (AMPA: NMDA) at ZT18 vs. ZT6. The number of PV cells and PV/PNN cells containing orthodenticle homeobox 2 (OTX2), which maintains PNNs, showed a strong trend toward an increase from ZT6 to ZT18. Diurnal fluctuations in PNNs and PV cells are expected to alter cortical excitatory:inhibitory balance and provide new insights into treatments for diseases impacted by disturbances in sleep and circadian rhythms.

Cocaine memory reactivation induces functional adaptations within parvalbumin interneurons in the rat medial prefrontal cortex.

Substance use disorder is a complex disease created in part by maladaptive learning and memory mechanisms following repeated drug use. Exposure to drug-associated stimuli engages prefrontal cortex circuits, and dysfunction of the medial prefrontal cortex (mPFC) is thought to underlie drug-seeking behaviors. Growing evidence supports a role for parvalbumin containing fast-spiking interneurons (FSI) in modulating prefrontal cortical microcircuit activity by influencing the balance of excitation and inhibition, which can influence learning and memory processes. Most parvalbumin FSIs within layer V of the prelimbic mPFC are surrounded by specialized extracellular matrix structures called perineuronal nets (PNN). Previous work by our group found that cocaine exposure altered PNN-surrounded FSI function, and pharmacological removal of PNNs reduced cocaine-seeking behavior. However, the role of FSIs and associated constituents (parvalbumin and PNNs) in cocaine-related memories was not previously explored and is still unknown. Here, we found that reactivation of a cocaine conditioned place preference memory produced changes in cortical PNN-surrounded parvalbumin FSIs, including decreased parvalbumin intensity, increased parvalbumin cell axis diameter, decreased intrinsic excitability, and increased excitatory synaptic input. Further investigation of intrinsic properties revealed changes in the interspike interval, membrane capacitance, and afterhyperpolarization recovery time. Changes in these specific properties suggest an increase in potassium-mediated currents, which was validated with additional electrophysiological analysis. Collectively, our results indicate that cocaine memory reactivation induces functional adaptations in PNN-surrounded parvalbumin neurons, which likely alters cortical output to promote cocaine-seeking behavior.

Sleep disruption elevates oxidative stress in parvalbumin-positive cells of the rat cerebral cortex.

We used a novel automated sleep disruption (SD) apparatus to determine the impact of SD on sleep and molecular markers of oxidative stress in parvalbumin (PV) neurons in the rat prefrontal cortex (PFC). Rats were subjected to two 6 hr SD sessions from zeitgeber time (ZT) 0 to ZT6, one by the gentle handling method and the other by an automated agitator running the length of the rat's home cage floor (a novel SD method). The same rats were later subjected to a 12 hr SD session from ZT0 to ZT12. Sleep was disrupted with both methods, although rats slept less during gentle handling than during the automated condition. Immediately after both SD sessions, rats displayed compensatory sleep characterized by elevated slow-wave activity. We measured in the prelimbic prefrontal cortex (prelimbic PFC; 6 and 12 hr SD) and orbital frontal cortex (12 hr SD) the intensity of the oxidative stress marker, 8-oxo-2'-deoxyguanosine (8-oxo-dG) as well as the staining intensity of PV and the PV cell-associated perineuronal net marker, Wisteria floribunda agglutinin (WFA). In the prelimbic PFC, 6 hr SD increased the intensity of 8-oxo-dG, PV, and WFA. After 12 hr SD, the intensity of 8-oxo-dG was elevated in all neurons. PV intensity was elevated only in neurons colabeled with 8-oxo-dG or WFA, and no changes were found in WFA intensity. We conclude that in association with SD-induced sleep drive, PV neurons in the prelimbic PFC exhibit oxidative stress.

Genetic Polymorphisms Affect Mouse and Human Trace Amine-Associated Receptor 1 Function.

Methamphetamine (MA) and neurotransmitter precursors and metabolites such as tyramine, octopamine, and ß-phenethylamine stimulate the G protein-coupled trace amine-associated receptor 1 (TAAR1). TAAR1 has been implicated in human conditions including obesity, schizophrenia, depression, fibromyalgia, migraine, and addiction. Additionally TAAR1 is expressed on lymphocytes and astrocytes involved in inflammation and response to infection. In brain, TAAR1 stimulation reduces synaptic dopamine availability and alters glutamatergic function. TAAR1 is also expressed at low levels in heart, and may regulate cardiovascular tone. Taar1 knockout mice orally self-administer more MA than wild type and are insensitive to its aversive effects. DBA/2J (D2) mice express a non-synonymous single nucleotide polymorphism (SNP) in Taar1 that does not respond to MA, and D2 mice are predisposed to high MA intake, compared to C57BL/6 (B6) mice. Here we demonstrate that endogenous agonists stimulate the recombinant B6 mouse TAAR1, but do not activate the D2 mouse receptor. Progeny of the B6XD2 (BxD) family of recombinant inbred (RI) strains have been used to characterize the genetic etiology of diseases, but contrary to expectations, BXDs derived 30-40 years ago express only the functional B6 Taar1 allele whereas some more recently derived BXD RI strains express the D2 allele. Data indicate that the D2 mutation arose subsequent to derivation of the original RIs. Finally, we demonstrate that SNPs in human TAAR1 alter its function, resulting in expressed, but functional, sub-functional and non-functional receptors. Our findings are important for identifying a predisposition to human diseases, as well as for developing personalized treatment options.

Extended exposure to environmental cues, but not to sucrose, reduces sucrose cue reactivity in rats.

In the present study, we examined the effects of extinction of sucrose-predictive contextual cues and/or sucrose satiation on the expression of sucrose cue reactivity in a rat model of relapse. Context extinction was imposed by housing rats in their home cage or in the operant conditioning chamber for 17 h prior to testing. For sucrose satiation, rats were allowed unlimited access to water or sucrose for 17 h prior to testing. Cue reactivity was assessed after either one (Day 1) or 30 (Day 30) days of forced abstinence from sucrose self-administration. An abstinence-dependent increase in sucrose cue reactivity was observed in all conditions ("incubation of craving"). Context extinction dramatically reduced lever responding on both Day 1 and Day 30. Sucrose satiation had no significant effect on cue reactivity in any condition. These results demonstrate that the context in which self-administration occurs maintains a powerful influence over cue reactivity, even after extended forced abstinence. In contrast, the primary reinforcer has little control over cue reactivity. These findings highlight the important role of conditioned contextual cues in driving relapse behavior.

A standardized and automated method of perineuronal net analysis using Wisteria floribunda agglutinin staining intensity.

Perineuronal nets (PNNs) are aggregations of extracellular matrix molecules that are critical for plasticity. Their altered development or changes during adulthood appear to contribute to a wide range of diseases/disorders of the brain. An increasing number of studies examining the contribution of PNN to various behaviors and types of plasticity have analyzed the fluorescence intensity of Wisteria floribunda agglutinin (WFA) as an indirect measure of the maturity of PNNs, with brighter WFA staining corresponding to a more mature PNN and dim WFA staining corresponding to an immature PNN. However, a clearly-defined and unified method for assessing the intensity of PNNs is critical to allow us to make comparisons across studies and to advance our understanding of how PNN plasticity contributes to normal brain function and brain disease states. Here we examined methods of PNN intensity quantification and demonstrate that creating a region of interest around each PNN and subtracting appropriate background is a viable method for PNN intensity quantification that can be automated. This method produces less variability and bias across experiments compared to other published analyses, and this method increases reproducibility and reliability of PNN intensity measures, which is critical for comparisons across studies in this emerging field.

Trace Amine-Associated Receptor 1 Regulation of Methamphetamine Intake and Related Traits.

Continued methamphetamine (MA) use is dependent on a positive MA experience and is likely attenuated by sensitivity to the aversive effects of MA. Bidirectional selective breeding of mice for high (MAHDR) or low (MALDR) voluntary consumption of MA demonstrates a genetic influence on MA intake. Quantitative trait locus (QTL) mapping identified a QTL on mouse chromosome 10 that accounts for greater than 50% of the genetically-determined differences in MA intake in the MAHDR and MALDR lines. The trace amine-associated receptor 1 gene (Taar1) is within the confidence interval of the QTL and encodes a receptor (TAAR1) that modulates monoamine neurotransmission and at which MA serves as an agonist. We demonstrate the existence of a non-functional allele of Taar1 in the DBA/2J mouse strain, one of the founder strains of the selected lines, and show that this non-functional allele co-segregates with high MA drinking and with reduced sensitivity to MA-induced conditioned taste aversion (CTA) and hypothermia. The functional Taar1 allele, derived from the other founder strain, C57BL/6J, segregates with low MA drinking and heightened sensitivity to MA-induced CTA and hypothermia. A role for TAAR1 in these phenotypes is corroborated in Taar1 transgenic mice: Taar1 knockout mice consume more MA and exhibit insensitivity to MA-induced CTA and hypothermia, compared with Taar1 wild-type mice. These are the first data to show that voluntary MA consumption is, in part, regulated by TAAR1 function. Behavioral and physiological studies indicate that TAAR1 function increases sensitivity to aversive effects of MA, and may thereby protect against MA use.

Effects of sodium butyrate on methamphetamine-sensitized locomotor activity.

Neuroadaptations associated with behavioral sensitization induced by repeated exposure to methamphetamine (MA) appear to be involved in compulsive drug pursuit and use. Increased histone acetylation, an epigenetic effect resulting in altered gene expression, may promote sensitized responses to psychostimulants. The role of histone acetylation in the expression and acquisition of MA-induced locomotor sensitization was examined by measuring the effect of histone deacetylase inhibition by sodium butyrate (NaB). For the effect on expression, mice were treated repeatedly with MA (10 days of 2mg/kg MA) or saline (10 days), and then vehicle or NaB (630 mg/kg, intraperitoneally) was administered 30 min prior to MA challenge and locomotor response was measured. NaB treatment increased the locomotor response to MA in both acutely MA treated and sensitized animals. For acquisition, NaB was administered 30 min prior to each MA exposure (10 days of 1 or 2mg/kg), but not prior to the MA challenge test. Treatment with NaB during the sensitization acquisition period significantly increased locomotor activation by MA in sensitized mice only. NaB alone did not significantly alter locomotor activity. Acute NaB or MA, but not the combination, increased striatal acetylation at histone H4. Repeated treatment with MA, but not NaB or MA plus NaB, increased striatal acetylation at histone H3. Although increased histone acetylation may alter the expression of genes involved in acute locomotor response to MA and in the acquisition of MA-induced sensitization, results for acetylation at H3 and H4 showed little correspondence with behavior.

Intermittent availability of ethanol does not always lead to elevated drinking in mice.

AIMS: Intermittent access (IA) to an alcohol (ethanol) solution can lead rats to higher ethanol intakes than continuous access, and a recent report showed increased drinking in C57BL/6J mice offered 20% ethanol vs. water 3X/week (Prior studies have offered ethanol during 24 h periods, either continuously or intermittently.). METHODS: We tested the high-preference C57BL/6J inbred mice: we also studied High Drinking in the Dark (HDID) mice, a line we have selectively bred to reach intoxicating blood ethanol levels after a short period of access to a single bottle of 20% ethanol. RESULTS: Neither HDID or C57BL/6J male mice offered ethanol every other day during only a 4-h access period showed greater daily intake than mice offered ethanol daily for 4 h. There was a small increase in drinking with 24 h IA in C57BL/6J mice. An experiment with HDID mice and their control heterogeneous stock stock modeled closely after a published study with C57BL/6J mice (Hwa, Chu, Levinson SA et al. Persistent escalation of alcohol drinking in C57BL/6J mice with intermittent access to 20% ethanol. Alcohol Clin Exp Res 2011;35:1938-1947) showed no significant elevation with 24 h IA exposure in either sex of any genotype. Finally, a near replication of the Hwa et al. study showed modestly greater intake in C57BL/6J mice, confirming the efficacy of 24 h IA. CONCLUSION: We conclude that 4 h of IA is likely insufficient to elevate drinking in mice. The lack of effect in HDID mice and their controls further suggests that not all genotypes respond to intermittency.

Effects of systemic or nucleus accumbens-directed dopamine D1 receptor antagonism on sucrose seeking in rats.

RATIONALE: Conditioned cues can elicit relapse to drug- and food-seeking behavior over prolonged periods of abstinence. If seeking behavior depends on mesolimbic dopamine D1 receptors, blocking these receptors should reduce seeking behavior. OBJECTIVES: We examined the effects of either systemic or intra-nucleus accumbens administration of the D1 antagonist SCH 23390 on extinction responding (sucrose seeking) by rats either 1 or 30 days into forced abstinence. MATERIALS AND METHODS: Rats self-administered 10% sucrose paired with a tone + light cue for 10 days. After either 1 or 30 days of forced abstinence, rats received systemic (0, 1, 5, or 25 µg/kg IP) or bilateral nucleus accumbens core or shell (0.3 or 0.6 µg/site) injections of SCH 23390 prior to extinction testing. RESULTS: Saline-treated rats responded more during extinction following 30 vs. 1 day of forced abstinence ("incubation of craving"). Systemic SCH 23390 reduced sucrose seeking after 1 day of forced abstinence, significantly reducing responding following pretreatment with 1, 5, and 25 µg/kg SCH 23390, but only 25 µg/kg significantly reduced sucrose seeking after 30 days of forced abstinence. SCH 23390 (0.3 or 0.6 µg/site) in the core or shell of the nucleus accumbens reduced sucrose seeking in all groups. CONCLUSION: Nucleus accumbens D1 receptors are involved in sucrose seeking, but it is not clear if they are involved in the incubation of craving. The fact that D1 antagonism reduced sucrose seeking across an extended period of abstinence may be of use for development of treatment strategies for relapse.

Abstinence-dependent transfer of lithium chloride-induced sucrose aversion to a sucrose-paired cue in rats.

RATIONALE: Responding for a drug- or sucrose-paired cue increases over forced abstinence (incubation of craving). If the incentive value of a cue depends on the incentive value of the primary reward, devaluing the primary reward should reduce cue reactivity. OBJECTIVES: We investigated whether conditioned taste aversion (CTA) to sucrose would transfer to a sucrose-paired cue after 1 or 30 days of forced abstinence and whether CTA after 1 day of forced abstinence would affect incubation of craving. MATERIALS AND METHODS: Rats self-administered 10% sucrose paired with a tone + light cue for 10 days. After 1 (Exp.1) or 30 (Exp.2) days of forced abstinence, rats received two home-cage pairings of sucrose with either LiCl (65 mg/kg, IP) to produce CTA or saline as a control. Two days later, rats responded for the cue alone. The following day, sucrose consumption was assessed in the same operant conditioning chamber. Exp.1 rats were tested again 1 month later to determine if CTA would affect incubation of craving. RESULTS: Exp.1: CTA after 1 day of forced abstinence did not attenuate cue reactivity when tested immediately after CTA, nor did the treatment affect incubation of craving or incubation of sucrose consumption. Exp.2: CTA after 1 month of forced abstinence resulted in a significant reduction in cue reactivity. CONCLUSION: The incentive values of sucrose and the conditioned representation of sucrose increase over an extended period of forced abstinence. This incubation appears to facilitate the transfer of an aversion to the primary reward to the conditioned cue.

Environmental enrichment attenuates cue-induced reinstatement of sucrose seeking in rats.

This study examined the effect of environmental enrichment on sucrose seeking in rats made abstinent from sucrose for 1 month, as measured by response for a tone+light cue previously associated with 10% sucrose self-administration. Rats were either enriched throughout the study (experiment 1) or only after sucrose self-administration training (experiment 2). Enrichment consisted of either housing the rats in pairs or grouping four rats (ENR4) in a large environment, both with novel objects. Controls (CON) were singly housed without novel objects. In experiment 1, ENR4 rats responded less to the sucrose-paired cue versus CON rats, but this difference was not statistically significant. In contrast, the decrease in response of ENR4 rats versus CON rats in experiment 2 was dramatic and significant. These findings, along with findings from other laboratories, support a hypothesis that the enrichment may provide individuals with a greater ability to discriminate the availability of reward. This may impart a decreased vulnerability to relapse behavior. Therefore, these results are relevant to both eating disorder and drug addiction - disorders characterized by relapse.