Adeno-associated Virus Expressing Cre Recombinase Research Articles

Knockdown of Tlr3 in dorsal striatum reduces ethanol consumption and acute functional tolerance in male mice

Systemic activation of toll-like receptor 3 (TLR3) signaling using poly(I:C), a TLR3 agonist, drives ethanol consumption in several rodent models, while global knockout of Tlr3 reduces drinking in C57BL/6J male mice. To determine if brain TLR3 pathways are involved in drinking behavior, we used CRISPR/Cas9 genome editing to generate a Tlr3 floxed (Tlr3F/F) mouse line. After sequence confirmation and functional validation of Tlr3 brain transcripts, we injected Tlr3F/F male mice with an adeno-associated virus expressing Cre recombinase (AAV5-CMV-Cre-GFP) to knockdown Tlr3 in the medial prefrontal cortex, nucleus accumbens, or dorsal striatum (DS). Only Tlr3 knockdown in the DS decreased two-bottle choice, every-other-day (2BC-EOD) ethanol consumption. DS-specific deletion of Tlr3 also increased intoxication and prevented acute functional tolerance to ethanol. In contrast, poly(I:C)-induced activation of TLR3 signaling decreased intoxication in male C57BL/6J mice, consistent with its ability to increase 2BC-EOD ethanol consumption in these mice. We also found that TLR3 was highly colocalized with DS neurons. AAV5-Cre transfection occurred predominantly in neurons, but there was minimal transfection in astrocytes and microglia. Collectively, our previous and current studies show that activating or inhibiting TLR3 signaling produces opposite effects on acute responses to ethanol and on ethanol consumption. While previous studies, however, used global knockout or systemic TLR3 activation (which alter peripheral and brain innate immune responses), the current results provide new evidence that brain TLR3 signaling regulates ethanol drinking. We propose that activation of TLR3 signaling in DS neurons increases ethanol consumption and that a striatal TLR3 pathway is a potential target to reduce excessive drinking.

Receptor Transporter Protein 4 (RTP4) in the Hypothalamus Is Involved in the Development of Antinociceptive Tolerance to Morphine.

Receptor transporter protein 4 (RTP4), one of the receptor chaperone proteins, contributes to the maturation and membrane trafficking of opioid receptor heteromers consisting of mu (MOPr) and delta (DOPr) opioid receptors (MOPr-DOPr). Although MOPr-DOPr is known to mediate the development of morphine tolerance, the extent to which RTP4 plays a role in this process has not been elucidated. Given that RTP4 can be upregulated by repeated administration of morphine, especially in the hypothalamus, here we investigated the effect of hypothalamus-selective ablation of RTP4 on the development of antinociceptive tolerance to morphine. In this study, we generated RTP4flox mice and selectively knocked-out RTP4 using local injection of adeno-associated virus expressing Cre recombinase (AAV-Cre) into the hypothalamus. The AAV-Cre injection partially, but significantly, decreased the level of RTP4 expression, and suppressed the development of antinociceptive tolerance to morphine. Next, we examined the mechanism of regulation of RTP4 and found that, in neuronal cells, Rtp4 induction is via Gi and MAPK activation, while, in microglial cells, the induction is via Toll-like receptor 4. Together, these studies highlight the role of MOR activity in regulating RTP4, which, in turn, plays an important role in modulating morphine effects in vivo.

196-OR: (Pro) renin Receptor Signaling in Tyrosine Hydroxylase Neurons in the Hypothalamic Paraventricular Nucleus Is Required for Obesity-Associated Glucose Metabolic Impairment

We recently reported that neuron-specific (pro) renin receptor (PRR) deletion reduces fasting blood glucose (BG) levels and improves glucose tolerance in high fat diet (HFD) -treated mice. To investigate underlying mechanisms, we focused on tyrosine hydroxylase (TH) neurons in the hypothalamic paraventricular nucleus (THPVN neurons) , a largely GABAergic population that expresses PRR and projects to autonomic centers important for BG regulation. To delete PRR from THPVN neurons, we injected into the PVN of PRR-loxp mice either an adeno-associated virus expressing cre recombinase under the control of the rat TH promoter (AAV2-TH-Cre) or AAV2-TH-eGFP as a control. After placing animals on a HFD for 6 wk, we found that the obesity-induced increase of fasting BG levels was blocked by PRR deletion from THPVN neurons (118.0 ± 3.7 vs. 139.3 ± 6.6 mg/dL, n=6/group, p= 0.04) , accompanied by a trend towards improved i.p. glucose tolerance (AUC: 12990 ± 1136 vs. 9467 ± 1298, n=6, p=0.07) . To further investigate the role of THPVN neurons in glucose homeostasis, we employed a Designer Receptors Exclusively Activated by Designer Drugs (DREADD) approach whereby AAV2-TH-Cre was injected bilaterally into the PVN of either hM3Dq-loxp (excitatory) or hM4Di-loxp (inhibitory) mice, and continuous glucose monitoring was performed in conscious, freely moving mice. Following i.p. injection of clozapine-N-oxide (CNO, 1 mg/Kg) or vehicle, we found that whereas basal BG levels were not altered by THPVN neuron activation, THPVN neuron inhibition significantly increased basal BG levels compared to vehicle in male (ΔBG: 31.6 ± 8.6 mg/dL vs. 11.0 ± 6.7 mg/dL, n=4, p=0.03) but not female mice. These findings establish a role for THPVN neuron PRR signaling in the pathogenesis of obesity-associated glucose metabolic impairment, and show that in male mice, basal THPVN neuron activity is required for normal glucose homeostasis. Disclosure L.Ac. souza: None. C.J.Worker: None. A.Gayban: None. M.W.Schwartz: None. Y.Feng earley: None. Funding National Institutes of Health (R01HL122770, R35HL155008, RDK83042, RDK124238)

Developmental, neurochemical, and behavioral analyses of ErbB4 Cyt-1 knockout mice.

Neuregulins (NRGs) and their cognate neuronal receptor ERBB4, which is expressed in GABAergic and dopaminergic neurons, regulate numerous behaviors in rodents and have been identified as schizophrenia at-risk genes. ErbB4 transcripts are alternatively spliced to generate isoforms that either include (Cyt-1) or exclude (Cyt-2) exon 26, which encodes a cytoplasmic domain that imparts ErbB4 receptors the ability to signal via the phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) pathway. Although ErbB4 Cyt-1/2 isoforms have been studied in transfected cultured cells, their functions in vivo remain unknown. Here, we generated ErbB4-floxed (ErbB4-Cyt1fl/fl ) mice to investigate the effects of germline (constitutive) and conditional (acute) deletions of the Cyt-1 exon. Overall receptor mRNA levels remain unchanged in germline ErbB4 Cyt-1 knockouts (Cyt-1 KOs), with all transcripts encoding Cyt-2 variants. In contrast to mice lacking all ErbB4 receptor function, GABAergic interneuron migration and number are unaltered in Cyt-1 KOs. However, basal extracellular dopamine (DA) levels in the medial prefrontal cortex are increased in Cyt-1 heterozygotes. Despite these neurochemical changes, Cyt-1 heterozygous and homozygous mice do not manifest behavioral abnormalities previously reported to be altered in ErbB4 null mice. To address the possibility that Cyt-2 variants compensate for the lack of Cyt-1 during development, we microinjected an adeno-associated virus expressing Cre-recombinase (AAV-Cre) into the DA-rich ventral tegmental area of adult ErbB4-Cyt1fl/fl mice to acutely target exon 26. These conditional Cyt-1 KOs were found to exhibit behavioral abnormalities in the elevated plus maze and startle response, consistent with the idea that late exon 26 ablations may circumvent compensation by Cyt-2 variants. Taken together, our observations indicate that ErbB4 Cyt-1 function in vivo is important for DA balance and behaviors in adults.

Chronic brain histamine depletion in adult mice induced depression-like behaviours and impaired sleep-wake cycle

Histamine acts as a neurotransmitter to regulate various physiological processes. Brain histamine is synthesized from an essential amino acid histidine in a reaction catalysed by histidine decarboxylase (Hdc). Hdc-positive neurons exist mainly in the tuberomammillary nucleus (TMN) of the posterior hypothalamus and project their axons to the entire brain. Recent studies have reported that a chronic decrease in histamine levels in the adult human brain was observed in several neurological disorders. However, it is poorly understood whether lower histamine levels play a causative role in those disorders. In the present study, we induced chronic histamine deficiency in the brains of adult mice to allow direct interpretation of the relationship between an impaired histaminergic nervous system and the resultant phenotype. To induce chronic brain histamine deficiency starting in adulthood, adeno-associated virus expressing Cre recombinase was microinjected into the TMN of Hdc flox mice (cKO mice) at the age of 8 weeks. Immunohistochemical analysis showed expression of Cre recombinase in the TMN of cKO mice. The reduction of histamine contents with the decreased Hdc expression in cKO brain was also confirmed. Behavioural studies revealed that chronic histamine depletion in cKO mice induced depression-like behaviour, decreased locomotor activity in the home cage, and impaired aversive memory. Sleep analysis showed that cKO mice exhibited a decrease in wakefulness and increase in non-rapid eye movement sleep throughout the day. Taken together, this study clearly demonstrates that chronic histamine depletion in the adult mouse brain plays a causative role in brain dysfunction.

Glycogen Synthase Kinase 3β in the Ventral Hippocampus is Important for Cocaine Reward and Object Location Memory

The ventral hippocampus is a component of the neural circuitry involved with context-associated memory for reward and generation of appropriate behavioral responses to context. Glycogen synthase kinase 3 beta (GSK3β) has been linked to the maintenance of synaptic plasticity, contextual memory retrieval, and is involved in the reconsolidation of cocaine-associated contextual memory. In this study, the effects of targeted downregulation of GSK3β in the ventral hippocampus were examined on a series of behavioral tests for assessing drug reward-context association and non-reward related memory. The Cre/loxP site-specific recombination system was used to knockdown GSK3β through bilateral stereotaxic delivery of an adeno-associated virus expressing Cre-recombinase (AAV-Cre) into the ventral hippocampus of adult mice homozygous for a floxed GSK3β allele. GSK3β floxed mice injected with AAV-Cre had a loss of 56–75% of GSK3β in the ventral hippocampus and displayed diminished development of cocaine conditioned place preference, but not morphine place preference as compared with wild-type mice injected with AAV-Cre or GSK3β floxed mice injected with a control virus, AAV-GFP. Impaired object location memory was observed in mice with GSK3β downregulation in the ventral hippocampus, but novel object recognition remained intact. These results indicate that GSK3β signaling in the ventral hippocampus is differentially involved in the formation of place-drug reward association dependent upon drug class. Additionally, ventral hippocampal GSK3β signaling is important in detection of discrete spatial cues, but not recognition memory for objects.

Conditional deletion of melanin-concentrating hormone receptor 1 from GABAergic neurons increases locomotor activity

ObjectiveMelanin-concentrating hormone (MCH) plays a key role in regulating energy balance. MCH acts via its receptor MCHR1, and MCHR1 deletion increases energy expenditure and locomotor activity, which is associated with a hyperdopaminergic state. Since MCHR1 expression is widespread, the neurons supporting the effects of MCH on energy expenditure are not clearly defined. There is a high density of MCHR1 neurons in the striatum, and these neurons are known to be GABAergic. We thus determined if MCH acts via this GABAergic neurocircuit to mediate energy balance. MethodsWe generated a Mchr1-flox mouse and crossed it with the Vgat-cre mouse to assess if MCHR1 deletion from GABAergic neurons expressing the vesicular GABA transporter (vGAT) in female Vgat-Mchr1-KO mice resulted in lower body weights or increased energy expenditure. Additionally, we determined if MCHR1-expressing neurons within the accumbens form part of the neural circuit underlying MCH-mediated energy balance by delivering an adeno-associated virus expressing Cre recombinase to the accumbens nucleus of Mchr1-flox mice. To evaluate if a dysregulated dopaminergic tone leads to their hyperactivity, we determined if the dopamine reuptake blocker GBR12909 prolonged the drug-induced locomotor activity in Vgat-Mchr1-KO mice. Furthermore, we also performed amperometry recordings to test whether MCHR1 deletion increases dopamine output within the accumbens and whether MCH can suppress dopamine release. ResultsVgat-Mchr1-KO mice have lower body weight, increased energy expenditure, and increased locomotor activity. Similarly, restricting MCHR1 deletion to the accumbens nucleus also increased locomotor activity. Vgat-Mchr1-KO mice show increased and prolonged sensitivity to GBR12909-induced locomotor activity, and amperometry recordings revealed that GBR12909 elevated accumbens dopamine levels to twice that of controls, thus MCHR1 deletion may lead to a hyperdopaminergic state that mediates their observed hyperactivity. Consistent with the inhibitory effect of MCH, we found that MCH acutely suppresses dopamine release within the accumbens. ConclusionsAs with established models of systemic MCH or MCHR1 deletion, we found that MCHR1 deletion from GABAergic neurons, specifically those within the accumbens nucleus, also led to increased locomotor activity. A hyperdopaminergic state underlies this increased locomotor activity, and is consistent with our finding that MCH signaling within the accumbens nucleus suppresses dopamine release. In effect, MCHR1 deletion may disinhibit dopamine release leading to the observed hyperactivity.

Regional knockdown of NDUFS4 implicates a thalamocortical circuit mediating anesthetic sensitivity.

Knockout of the mitochondrial complex I protein, NDUFS4, profoundly increases sensitivity of mice to volatile anesthetics. In mice carrying an Ndufs4lox/lox gene, adeno-associated virus expressing Cre recombinase was injected into regions of the brain postulated to affect sensitivity to volatile anesthetics. These injections generated otherwise phenotypically wild type mice with region-specific, postnatal inactivation of Ndufs4, minimizing developmental effects of gene loss. Sensitivities to the volatile anesthetics isoflurane and halothane were measured using loss of righting reflex (LORR) and movement in response to tail clamp (TC) as endpoints. Knockdown (KD) of Ndufs4 in the vestibular nucleus produced resistance to both anesthetics for movement in response to TC. Ndufs4 loss in the central and dorsal medial thalami and in the parietal association cortex increased anesthetic sensitivity to both TC and LORR. Knockdown of Ndufs4 only in the parietal association cortex produced striking hypersensitivity for both endpoints, and accounted for half the total change seen in the global KO (Ndufs4(KO)). Excitatory synaptic transmission in the parietal association cortex in slices from Ndufs4(KO) animals was hypersensitive to isoflurane compared to control slices. We identified a direct neural circuit between the parietal association cortex and the central thalamus, consistent with a model in which isoflurane sensitivity is mediated by a thalamic signal relayed through excitatory synapses to the parietal association cortex. We postulate that the thalamocortical circuit is crucial for maintenance of consciousness and is disrupted by the inhibitory effects of isoflurane/halothane on mitochondria.

Adult Brain Serotonin Deficiency Causes Hyperactivity, Circadian Disruption, and Elimination of Siestas.

Serotonin (5-HT) is a crucial neuromodulator linked to many psychiatric disorders. However, after more than 60 years of study, its role in behavior remains poorly understood, in part because of a lack of methods to target 5-HT synthesis specifically in the adult brain. Here, we have developed a genetic approach that reproducibly achieves near-complete elimination of 5-HT synthesis from the adult ascending 5-HT system by stereotaxic injection of an adeno-associated virus expressing Cre recombinase (AAV-Cre) into the midbrain/pons of mice carrying a loxP-conditional tryptophan hydroxylase 2 (Tph2) allele. We investigated the behavioral effects of deficient brain 5-HT synthesis and discovered a unique composite phenotype. Surprisingly, adult 5-HT deficiency did not affect anxiety-like behavior, but resulted in a robust hyperactivity phenotype in novel and home cage environments. Moreover, loss of 5-HT led to an altered pattern of circadian behavior characterized by an advance in the onset and a delay in the offset of daily activity, thus revealing a requirement for adult 5-HT in the control of daily activity patterns. Notably, after normalizing for hyperactivity, we found that the normal prolonged break in nocturnal activity (siesta), a period of rapid eye movement (REM) and non-REM sleep, was absent in all animals in which 5-HT deficiency was verified. Our findings identify adult 5-HT as a requirement for siestas, implicate adult 5-HT in sleep-wake homeostasis, and highlight the importance of our adult-specific 5-HT-synthesis-targeting approach in understanding 5-HT's role in controlling behavior. Serotonin (5-HT) is a crucial neuromodulator, yet its role in behavior remains poorly understood, in part because of a lack of methods to target specifically adult brain 5-HT synthesis. We developed an approach that reproducibly achieves near-complete elimination of 5-HT synthesis from the adult ascending 5-HT system. Using this technique, we discovered that adult 5-HT deficiency led to a novel compound phenotype consisting of hyperactivity, disrupted circadian behavior patterns, and elimination of siestas, a period of increased sleep during the active phase. These findings highlight the importance of our approach in understanding 5-HT's role in behavior, especially in controlling activity levels, circadian behavior, and sleep-wake homeostasis, behaviors that are disrupted in many psychiatric disorders such as attention deficit hyperactivity disorder.

Selective deletion of leptin receptors in adult hippocampus induces depression-related behaviours

Previous studies have demonstrated that leptin and its receptors (LepRb) in the central nervous system play an important role in regulating depression- and anxiety-related behaviours. However, the physiological functions of LepRb in specific brain regions for mediating different emotional behaviours remain to be defined. In this study, we examined the behavioural effects of LepRb ablation in the adult hippocampus using a series of behavioural paradigms for assessing depression- and anxiety-related behaviours. Targeted deletion of LepRb was achieved using the Cre/loxP site-specific recombination system through bilateral stereotaxic delivery of an adeno-associated virus expressing Cre-recombinase (AAV-Cre) into the dentate gyrus of adult mice homozygous for a floxed leptin receptor allele. AAV-Cre-mediated deletion of the floxed region of LepRb was detected 2 wk after injection. In accordance with this, leptin-stimulated phosphorylation of Akt was attenuated in the hippocampus of AAV-Cre injected mice. Mice injected with AAV-Cre displayed normal locomotor activity and anxiety-like behaviour, as determined in the elevated plus-maze, light-dark box and open field tests, but showed increased depression-like behaviours in the tail suspension, saccharin preference and learned helplessness tests. Taken together, these data suggest that deletion of LepRb in the adult hippocampus is sufficient to induce depression-like behaviours. Our results support the view that leptin signalling in the hippocampus may be essential for positive mood states and active coping to stress.

HDAC3 Is a Negative Regulator of Cocaine-Context-Associated Memory Formation

Cocaine-induced neuroplasticity mediated by histone acetylating and deacetylating enzymes may contribute to addiction-like behaviors. For example, overexpression of histone deacetylases (HDACs) 4 or 5 in the nucleus accumbens suppresses cocaine-induced conditioned place preference (CPP) acquisition in mice. HDAC4 and HDAC5 are known to interact with HDAC3, but the role of HDAC3 in cocaine-induced behaviors has never been examined. In this study, we address the hypothesis that HDAC3 is a negative regulator of cocaine-context-associated memory formation in mice. We examined the role of HDAC3 during the conditioning phase of CPP, when the mouse has the opportunity to form an associative memory between the cocaine-paired context and the subjective effects of cocaine. To address this hypothesis, Hdac3(flox/flox) and Hdac3(+/+) mice (generated from a C57BL/6 background) were infused into the nucleus accumbens with adeno-associated virus expressing Cre recombinase to create focal, homozygous Hdac3 deletions. Hdac3(flox/flox) mice exhibit significantly enhanced CPP acquisition, which is correlated with increased gene expression during the consolidation phase of acquisition. Increased gene expression of c-Fos and Nr4a2 is correlated with decreased HDAC3 occupancy and increased histone H4 lysine 8 acetylation at their promoters. The results from this study demonstrate that HDAC3 negatively regulates cocaine-induced CPP acquisition.

Running‐induced anxiety is dependent on increases in hippocampal neurogenesis

Exercise, specifically voluntary wheel running, is a potent stimulator of hippocampal neurogenesis in adult mice. In addition, exercise induces behavioral changes in numerous measures of anxiety in rodents. However, the physiological underpinnings of these changes are poorly understood. To investigate the role of neurogenesis in exercise-mediated anxiety, we examined the cellular and behavioral effects of voluntary wheel running in mice with a reduction in hippocampal neurogenesis, achieved through conditional deletion of ataxia telangiectasia-mutated and rad-3-related protein (ATR), a cell cycle checkpoint kinase necessary for normal levels of neurogenesis. Following hippocampal microinjection of an adeno-associated virus expressing Cre recombinase to delete ATR, mice were exposed to 4 weeks of voluntary wheel running and subsequently evaluated for anxiety-like behavior. Wheel running resulted in increased cell proliferation and neurogenesis, as measured by bromodeoxyuridine and doublecortin, respectively. Wheel running also resulted in heightened anxiety in the novelty-induced hypophagia, open field and light-dark box tests. However, both the neurogenic and anxiogenic effects of wheel running were attenuated following hippocampal ATR deletion, suggesting that increased neurogenesis is an important mediator of exercise-induced anxiety.

HIF‐1a gene deletion in the nucleus tractus solitarii (NTS) blunts ventilatory acclimatization to hypoxia (VAH)

Previously, we found that Hypoxia‐Inducible Factor‐1α (HIF‐1α) expression in the central nervous system (CNS) is necessary for normal VAH in mice. Here we tested the hypothesis that HIF‐1α in the NTS, which receives sensory input from carotid body chemoreceptors, is necessary during hypoxic exposure for normal VAH. We used the Cre‐LoxP strategy and microinjected Adeno‐associated virus expressing Cre‐recombinase (AAV‐Cre) into the NTS of mice carrying conditional HIF‐1α floxed alleles. AAV‐LacZ was microinjected as a control and to determine the extent of AAV expression. One week after microinjection, mice were kept in normoxia (CN) or CH (Pio2 = 70 Torr) for 7 days before measuring the hypoxic ventilatory response with barometric pressure plethysmography. Ventilation (Vi mL/(min kg)) was: CHRONIC NORMOXIA Inspired O2 (%) AAV‐Cre (n=6) AAV‐LacZ (n=6) 21 2176 ± 158 2059 ± 106 10 2088 ± 181 1905 ± 84 CHRONIC HYPOXIA Inspired O2 (%) AAV‐Cre (n=9) AAV‐LacZ (n=8) 21 2811 ± 121 2949 ± 152 10 3092 ± 245 4566 ± 307 CH significantly increased Vi as expected, but the increases in Vi and breathing frequency in CH were significantly less with AAV‐Cre than with AAV‐LacZ. The results support HIF‐1α in the NTS contributing to the increased CNS gain of the HVR in VAH we reported previously (J. Appl. Physiol. 87: 817–823, 1999). Supported by NIH RO1 HL‐081823 and 1P01 HL 098053.

Compensatory Regulation of Dopamine after Ablation of the Tyrosine Hydroxylase Gene in the Nigrostriatal Projection

The tyrosine hydroxylase (TH; EC 1.14.16.2) is a rate-limiting enzyme in the dopamine synthesis and important for the central dopaminergic system, which controls voluntary movements and reward-dependent behaviors. Here, to further explore the regulatory mechanism of dopamine levels by TH in adult mouse brains, we employed a genetic method to inactivate the Th gene in the nigrostriatal projection using the Cre-loxP system. Stereotaxic injection of adeno-associated virus expressing Cre recombinase (AAV-Cre) into the substantia nigra pars compacta (SNc), where dopaminergic cell bodies locate, specifically inactivated the Th gene. Whereas the number of TH-expressing cells decreased to less than 40% in the SNc 2 weeks after the AAV-Cre injection, the striatal TH protein level decreased to 75%, 50%, and 39% at 2, 4, and 8 weeks, respectively, after the injection. Thus, unexpectedly, the reduction of TH protein in the striatum, where SNc dopaminergic axons innervate densely, was slower than in the SNc. Moreover, despite the essential requirement of TH for dopamine synthesis, the striatal dopamine contents were only moderately decreased, to 70% even 8 weeks after AAV-Cre injection. Concurrently, in vivo synthesis activity of l-dihydroxyphenylalanine, the dopamine precursor, per TH protein level was augmented, suggesting up-regulation of dopamine synthesis activity in the intact nigrostriatal axons. Collectively, our conditional Th gene targeting method demonstrates two regulatory mechanisms of TH in axon terminals for dopamine homeostasis in vivo: local regulation of TH protein amount independent of soma and trans-axonal regulation of apparent L-dihydroxyphenylalanine synthesis activity per TH protein.

HDAC3 Is a Critical Negative Regulator of Long-Term Memory Formation

Gene expression is dynamically regulated by chromatin modifications on histone tails, such as acetylation. In general, histone acetylation promotes transcription, whereas histone deacetylation negatively regulates transcription. The interplay between histone acetyltranserases and histone deacetylases (HDACs) is pivotal for the regulation of gene expression required for long-term memory processes. Currently, very little is known about the role of individual HDACs in learning and memory. We examined the role of HDAC3 in long-term memory using a combined genetic and pharmacologic approach. We used HDAC3-FLOX genetically modified mice in combination with adeno-associated virus-expressing Cre recombinase to generate focal homozygous deletions of Hdac3 in area CA1 of the dorsal hippocampus. To complement this approach, we also used a selective inhibitor of HDAC3, RGFP136 [N-(6-(2-amino-4-fluorophenylamino)-6-oxohexyl)-4-methylbenzamide]. Immunohistochemistry showed that focal deletion or intrahippocampal delivery of RGFP136 resulted in increased histone acetylation. Both the focal deletion of HDAC3 as well as HDAC3 inhibition via RGFP136 significantly enhanced long-term memory in a persistent manner. Next we examined expression of genes implicated in long-term memory from dorsal hippocampal punches using quantitative reverse transcription-PCR. Expression of nuclear receptor subfamily 4 group A, member 2 (Nr4a2) and c-fos was significantly increased in the hippocampus of HDAC3-FLOX mice compared with wild-type controls. Memory enhancements observed in HDAC3-FLOX mice were abolished by intrahippocampal delivery of Nr4a2 small interfering RNA, suggesting a mechanism by which HDAC3 negatively regulates memory formation. Together, these findings demonstrate a critical role for HDAC3 in the molecular mechanisms underlying long-term memory formation.

HDAC3 is a critical negative regulator of long-term memory formation

Gene expression is dynamically regulated by chromatin modifications on histone tails, such as acetylation. In general, histone acetylation promotes transcription, whereas histone deacetylation negatively regulates transcription. The interplay between histone acetyltranserases and histone deacetylases (HDACs) is pivotal for the regulation of gene expression required for long-term memory processes. Currently, very little is known about the role of individual HDACs in learning and memory. We examined the role of HDAC3 in long-term memory using a combined genetic and pharmacologic approach. We used HDAC3–FLOX genetically modified mice in combination with adeno-associated virus-expressing Cre recombinase to generate focal homozygous deletions of Hdac3 in area CA1 of the dorsal hippocampus. To complement this approach, we also used a selective inhibitor of HDAC3, RGFP136 [N-(6-(2-amino-4-fluorophenylamino)-6-oxohexyl)-4-methylbenzamide]. Immunohistochemistry showed that focal deletion or intrahippocampal delivery of RGFP136 resulted in increased histone acetylation. Both the focal deletion of HDAC3 as well as HDAC3 inhibition via RGFP136 significantly enhanced long-term memory in a persistent manner. Next we examined expression of genes implicated in long-term memory from dorsal hippocampal punches using quantitative reverse transcription-PCR. Expression of nuclear receptor subfamily 4 group A, member 2 (Nr4a2) and c-fos was significantly increased in the hippocampus of HDAC3–FLOX mice compared with wild-type controls. Memory enhancements observed in HDAC3–FLOX mice were abolished by intrahippocampal delivery of Nr4a2 small interfering RNA, suggesting a mechanism by which HDAC3 negatively regulates memory formation. Together, these findings demonstrate a critical role for HDAC3 in the molecular mechanisms underlying long-term memory formation.

Doxycycline treatment prevents alveolar destruction in VEGF‐deficient mouse lung

In vivo lung-targeted VEGF gene inactivation results in pulmonary cell apoptosis, airspace enlargement, and increased lung compliance consistent with an emphysema-like phenotype. The predominant hypothesis for the cause of lung destruction in emphysema is an imbalance between active lung protease and anti-protease molecules. Therefore, we investigated the role of protease (e.g., matrix metalloproteinases--MMPs) and anti-protease (e.g., tissue inhibitors of metalloproteinases--TIMPs) expression in contributing to the lung structural remodeling observed in pulmonary-VEGF-deficient mice. VEGFLoxP mice instilled through the trachea with an adeno-associated virus expressing Cre recombinase (AAV/Cre) manifest airspace enlargement and a greater (P < 0.05) mean linear intercept (MLI: 44.2 +/- 4.2 microm) compared to mice instilled with a control virus expressing LacZ (31.3 +/- 2.5 microm). Airspace enlargement was prevented by the continuous administration of the general MMP inhibitor, doxycycline (Dox) (Cre + Dox: 32.6 +/- 2.5 microm), and MLI values were not different from either control (LacZ + Dox: 30.5 +/- 1.2 microm). In situ magnetic resonance imaging of VEGF gene inactivated mouse lungs revealed uneven inflation, residual trapped gas volumes upon oxygen absorption deflation/re-inflation, and loss of parenchymal structure; effects that were largely prevented by Dox. Five weeks after AAV/Cre infection Western blot revealed a 9.9-fold increase in pulmonary MMP-3, and 2-fold increases in MMP-9 and TIMP-2. However, the increase in MMP-3 was prevented by Dox administration and was associated with a 2-fold increase in serpin b5 (Maspin) expression. These results suggest that doxycycline treatment largely prevents the aberrant lung remodeling response observed in VEGF-deficient mouse lungs and is associated with changes in protease and anti-protease expression.

A Conditional Deletion of the NR1 Subunit of the NMDA Receptor in Adult Spinal Cord Dorsal Horn Reduces NMDA Currents and Injury-Induced Pain

To determine the importance of the NMDA receptor (NMDAR) in pain hypersensitivity after injury, the NMDAR1 (NR1) subunit was selectively deleted in the lumbar spinal cord of adult mice by the localized injection of an adenoassociated virus expressing Cre recombinase into floxed NR1 mice. NR1 subunit mRNA and dendritic protein are reduced by 80% in the area of the virus injection, and NMDA currents, but not AMPA currents, are reduced 86-88% in lamina II neurons. The spatial NR1 knock-out does not alter heat or cold paw-withdrawal latencies, mechanical threshold, or motor function. However, injury-induced pain produced by intraplantar formalin is reduced by 70%. Our results demonstrate conclusively that the postsynaptic NR1 receptor subunit in the lumbar dorsal horn of the spinal cord is required for central sensitization, the central facilitation of pain transmission produced by peripheral injury.