Regulation of 5-HT2A/C receptors and DOI-induced behaviors by protein kinase Cγ

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Abstract

Protein kinase Cγ (PKCγ) null mutant mice demonstrate increased behavioral impulsivity and ethanol consumption. Pharmacological studies have shown that 5-HT2A/C receptors modulate impulsivity and ethanol consumption in rodents and that PKC can regulate 5-HT2A/C receptors. To determine whether PKCγ plays a selective role in 5-HT2A/C receptor regulation, biochemical and behavioral experiments were performed in PKCγ mutant and wild-type mice. DOI-stimulated phosphoinositol hydrolysis and [125I]-DOI saturation binding in the PFC, and quantitative autoradiography of [125I]-DOI binding sites in 15 brain regions were analyzed. DOI-induced head twitch responses (HTR) were measured in naive mice after an acute 2.5 mg/kg injection of DOI. Results indicated that DOI-induced HTR was significantly greater in mutant mice compared to wild-type mice. Results of the phosphoinositol hydrolysis, membrane binding, and autoradiography experiments indicated that in mutant mice, increased HTR was associated with increased 5-HT2A/C receptor function in the PFC, but not increased receptor number or affinity suggesting that PKCγ regulates receptor function but not receptor number. These data support a role for 5-HT2A/C receptors in the PFC in mediating some of the behavioral differences observed between PKCγ mutant and wild-type mice.

Introduction

Null mutant mice lacking the neuronal-specific gamma isotype of protein kinase C (PKCγ) are more impulsive in an appetitive signaled-nosepoke task and consume more ethanol in a free-choice paradigm when compared to wild-type control mice (Bowers and Wehner, 2001). The neural mechanism by which PKCγ impacts these behaviors is most likely complex. However, PKCγ is primarily located post-synaptically; therefore, it is possible to limit the investigation of mechanisms to post-synaptic processes. A logical neurotransmitter candidate is the serotonin (5-HT) system, as numerous human and animal studies have shown that dysfunction of the central serotonergic system is related to both increased behavioral disinhibition (Harrison et al., 1997, Harrison et al., 1999, Ho et al., 1998, Leyton et al., 2001) and increased ethanol consumption (Fils-Aime et al., 1996, LeMarquand et al., 1994a, LeMarquand et al., 1994b, McBride et al., 1995, Murphy et al., 1982).

The 5-HT receptor system is heterogeneous with at least 15 subpopulations of neuronal receptors from 7 distinct families (5-HT1–5-HT7), which are encoded by different genes (Barnes and Sharp, 1999). One of these families, 5-HT2A/B/C, has been shown to be associated with PKC such that agonist-stimulated receptor desensitization and down-regulation are regulated by PKC activity (Anji et al., 2001, Kagaya et al., 1990, Roth et al., 1998). A recent investigation of PKC isotype expression in neonatal rat spinal cord after 5-HT2 receptor stimulation reported that gene expression for the PKCγ isotype was upregulated after treatment with the 5-HT2A/C agonist, 2-5-dimethoxy-4-iodoamphetamine (DOI) (MacDonald et al., 2001). In addition, Anji et al. (2001) implicate PKCα and PKCγ in the regulation of 5-HT2A receptor mRNA. This suggests a possible selective role of PKCγ in 5-HT2 receptor regulation. Further support for this association is indicated by the shared brain regional localization of PKCγ and 5-HT2 receptors, including prefrontal cortex, as well as their post-synaptic localizations (Backstrom et al., 2001, Naik et al., 2000, Pazos et al., 1985, Thomas and Everitt, 2001).

In the present study, experiments were designed to explore further the potential link between PKCγ, 5-HT2A/C receptors, prefrontal cortex (PFC), and altered behavioral responses in PKCγ mutants compared to wild-type mice. To test the hypothesis that PKCγ null mutant mice have a dysregulation in the function and/or number of 5-HT2A/C receptors in the PFC, we evaluated DOI-stimulated phosphoinositol (PI) hydrolysis in slices isolated from the PFC of mutant and wild-type mice. In addition, we measured [125I]-DOI binding using tissue extracts from the PFC followed by quantitative autoradiography of [125I]-DOI binding sites in several brain regions including the PFC and other regions associated with drug reward. To confirm the role of 5-HT2A/C receptors in regulating behavioral differences between PKCγ mutant and wild-type mice, we extended our previous studies of behavior to include a characterization of DOI-induced head twitch responses (HTR) because this response is regulated by the serotonin system in the PFC (Willins and Meltzer, 1997). The head twitch response is a behavioral manifestation of the functional activation of 5-HT2 receptors (Darmani and Gerdes, 1995, Darmani et al., 1990, Darmani et al., 1992, Willins and Meltzer, 1997) and can be measured easily and quickly in mice.

Section snippets

Animals

Male and female mice were 80–120 days of age at the time of testing and were housed in like-sex groups of 2–5. Mice were given food and water ad libitum and maintained on a 12 h light/dark cycle (lights on at 7:00 AM). PKCγ null mutant mice were derived using gene-targeting and homologous recombination techniques (Abeliovich et al., 1993) and are currently bred on an F1 C57BL/6 X 129/S6 mixed genetic background at the Institute for Behavioral Genetics (Boulder, CO). The F1 generations are bred

Phosphoinositol hydrolysis

Fig. 1 shows a comparison of 10 μM DOI-stimulated phosphoinositol hydrolysis in the prefrontal cortices of mutant and wild-type mice indicating that 5-HT2A/C function was significantly greater in mutant mice (t14 = 2.58, p < 0.02). DOI-stimulation over basal values averaged 125% in mutant PFC compared 108% in wild-type PFC.

[125I]-DOI binding

Studies using membrane preparations from prefrontal cortex were performed to determine Bmax and Kd values. Results from the non-linear regression analysis of saturation binding

Discussion

The primary objective of the present study was to determine whether the neuronal-specific isotype of PKC, PKCγ, regulates 5-HT2A/C receptor number and function in the prefrontal cortex and whether there are behavioral correlates relating to this potential dysfunction. In support of this hypothesis the relevant findings of this study are: 1) in naive PKCγ null mutant mice 5-HT2A/C receptor function, as measured by phosphoinositol hydrolysis, is increased in prefrontal cortex compared to

Acknowledgements

This work was supported by NIH grants, AA13901 to BJB; and AA03527 and AA13018 to JMW.

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