AbstractGiven the complexity of the interaction of the physiological, genetic and environmental factors driving the clinical expression of bipolar disorder phenotypes, its neurobiology is yet to be unraveled and avenues to new pharmacotherapies are just beginning to be explored. The present chapter describes the latest findings from studies of the mechanism of action of our most effective treatments, i.e., lithium and anticonvulsants, as well as ongoing multidisciplinary research in this field utilizing not only genetic, neuroimaging, and peripheral biological markers in patient populations but also postmortem neuroanatomical and neurochemical approaches. The data to‐date do not provide a comprehensive description of the pathophysiology of bipolar disorder or the mechanisms for mood‐stabilization. Enhanced understanding of the long‐term prophylactic action of drugs like lithium in the brain and current advances in the combined use of neuroimaging and genetics will lead to important insights into the neurobiology of the disease and to a rational basis for the development of the novel antibipolar drugs of the future.

The myristoylated alanine‐rich C kinase substrate (MARCKS) is a primary protein kinase C (PKC) substrate in brain thought to transduce PKC signaling into alterations in the filamentous (F) actin cytoskeleton. Within the adult hippocampus, MARCKS is highly expressed in the dentate gyrus (DG)‐CA3 mossy fiber pathway, but is expressed at low levels in the CA3‐CA1 Schaffer collateral‐CA1 pathway. We have previously demonstrated that 50% reductions in MARCKS expression in heterozygous Marcks mutant mice produce robust deficits in spatial reversal learning, but not contextual fear conditioning, suggesting that only specific aspects of hippocampal function are impaired by reduction in MARCKS expression. To further elucidate the role of MARCKS in hippocampal synaptic plasticity, in the present study we examined basal synaptic transmission, paired‐pulse facilitation, post‐tetanic potentiation, and long‐term potentiation (LTP) in the hippocampal mossy fiber‐CA3 and Schaffer collateral‐CA1 pathways of heterozygous Marcks mutant and wild‐type mice. We found that LTP is significantly impaired in the mossy fiber‐CA3 pathway, but not in the Schaffer collateral‐CA1 pathway, in heterozygous Marcks mutant mice, whereas basal synaptic transmission, paired‐pulse facilitation, and post‐tetanic potentiation are unaffected in both pathways. These findings indicate that a 50% reduction in MARCKS expression impairs processes required for long‐term, but not short‐term, synaptic plasticity in the mossy fiber‐CA3 pathway. The implications of these findings for the role of the mossy fiber‐CA3 pathway in hippocampus‐dependent learning processes are discussed. © 2006 Wiley‐Liss Inc.

The myristoylated alanine-rich C kinase substrate (MARCKS) is a primary substrate of protein kinase C (PKC) thought to regulate membrane-filamentous actin cytoskeletal plasticity in response to PKC activity in the regulation of synaptic efficacy. We have recently reported that MARCKS expression is significantly elevated (45%) in the hippocampus of DBA/2J mice, which exhibit impaired hippocampus-dependent learning and hippocampal long-term potentiation (LTP), compared with C57BL/6J mice. The latter finding led us to hypothesize that elevations in MARCKS expression are detrimental to hippocampal plasticity and function. To assess this more directly, we examined hippocampal (CA1) paired-pulse facilitation and LTP, and hippocampus-dependent learning in mice overexpressing MARCKS through the expression of a human MARCKS transgene (Tg(+)). The human MARCKS protein was confirmed to be expressed in the hippocampus of Tg(+) mice but not in Tg(-) mice. Schaffer collateral paired-pulse facilitation, input-output responses, and LTP did not differ between Tg+ and Tg(-) mice, indicating that neurotransmitter release, short-term, and long-term synaptic plasticity are not impaired by MARCKS overexpression. In the Morris water maze, Tg(+) mice exhibited a mild but significant spatial learning impairment during initial acquisition, and a more severe impairment during reversal training. Tg(+) did not exhibit impaired swim speed or visible platform performance relative to Tg(-) mice, indicating the absence of gross sensorimotor deficits. Fear conditioning to either context or cue was not impaired in Tg(+) mice. Behavioral deficits could not be attributed to differences in hippocampal PKC isozyme (alpha, beta(II), gamma, epsilon, zeta) or calmodulin expression, or alterations in hippocampal cytoarchitecture or infrapyramidal mossy fiber limb length. Collectively, these results indicate that elevations in MARCKS expression are detrimental to specific aspects of hippocampal function. (c) 2005 Wiley-Liss, Inc.

The clinical efficacy of lithium to prophylactically reduce profound mood cycling inpatients with bipolar disorder is indicative of its ability to stabilize the underlying disease mechanism(s). Chronic lithium may exert its therapeutic effects by normalizing aberrant phosphoinositide signaling through its interactions with the phosphoinositide pathway and down-stream alterations in protein kinase C (PKC) activity and substrate phosphorylation. We have shown that chronic, but not acute, lithium treatment at clinically relevant concentrations significantly reduces the expression of MARCKS (myristoylated alanine rich C-kinase substrate), a primary and preferential PKC substrate, and that MARCKS gene expression is regulated by chronic lithium at the transcriptional level through a lithium-responsive element in the MARCKS promoter. The MARCKS protein is located in pre- and postsynaptic terminals where it has been implicated to regulate phosphoinositide hydrolysis, filamentous-actin cytoskeletal plasticity, vesicular trafficking and the release efficacy of several neurotransmitters. MARCKS is necessary for normal mouse brain development, and remains highly expressed in several key adult cortical, limbic, and subcortical brain regions. Several neuroplastic events are sensitive to alterations in MARCKS expression, and prolonged elevations in PKC activity, physiological stress, and proinflammatory factors induce alterations in MARCKS expression. The long-term mood-stabilizing effect, as well as some side-effects (memory impairment), of chronic lithium may stem from its capacity to down-regulate MARCKS expression and/or phosphorylation in dysregulated circuits of key brain regions.

Auditory evoked potentials have been used in a variety of animal models to assess information-processing impairments in schizophrenia. Previous mouse models have primarily employed a paired click paradigm to assess the transient measures of auditory gating. The current study uses stimulus trains at varied interstimulus intervals (ISI) between 0.25 and 8 s in mice to assess the effects of chronic olanzapine and haloperidol on auditory processing. Data indicate that olanzapine increases the amplitude of the N40, P80, and P20/N40 components of the auditory evoked potential, whereas haloperidol had no such effect. The ISI paradigm also allowed for an evaluation of several components of the mouse evoked potential to assess those that display response properties similar to the human P50 and N100. Data suggest that the mouse N40 displays an ISI response relationship that shares characteristics with the human N100, whereas the P20 appears more consistent with the human P50 across the ISI range evaluated in this task. This study suggests that olanzapine may help improve N100 impairments seen in schizophrenia, while haloperidol does not.

Protein kinase Cγ (PKCγ) is highly expressed in the rodent hippocampus and has been implicated in long-term alterations in synaptic efficacy. Acute stress has been shown to negatively affect hippocampal synaptic plasticity, and the present study examined the effect of acute stress on PKCγ expression/subcellular distribution by quantitative western blotting in two inbred mouse strains (C57BL/6J versus DBA/2J) with established differences in hippocampal plasticity. It was found that both DBA/2J and C57BL/6J strains exhibited similar basal, stress-induced elevations, and recovery of serum corticosterone levels. Acute stress produced a significant reduction in both membrane and cytosolic PKCγ expression in the hippocampus of C57BL/6J mice compared to no-stress controls, but did not alter either membrane or cytosolic PKCγ expression in the hippocampus of DBA/2J mice compared to no-stress controls. These data provide direct evidence that PKCγ is differentially regulated in the hippocampus of C57BL/6J and DBA/2J mice by acute stress. The role of stress-induced regulation of hippocampal PKCγ expression in hippocampal synaptic plasticity is discussed.

The myristoylated alanine‐rich C kinase substrate (MARCKS) is a major protein kinase C (PKC) substrate in brain that binds the inner surface of the plasma membrane, calmodulin, and cross‐links filamentous actin, all in a PKC phosphorylation‐reversible manner. MARCKS has been implicated in hippocampal‐dependent learning and long‐term potentiation (LTP). Previous studies have shown DBA/2 mice to exhibit poor spatial/contextual learning, impaired hippocampal LTP, and hippocampal mossy fiber hypoplasia, as well as reduced hippocampal PKC activity and expression relative to C57BL/6 mice. In the present study, we assessed the expression (mRNA and protein) and subcellular distribution (membrane and cytolsol) of MARCKS in the hippocampus and frontal cortex of C57BL/6 and DBA/2 mice using quantitative western blotting. In the hippocampus, total MARCKS mRNA and protein levels in C57BL/6J mice were significantly lower (∼45%) compared with DBA/2J mice, and MARCKS protein was observed predominantly in the cytosolic fraction. MARCKS expression in frontal cortex did not differ significantly between strains. To examine the dynamic regulation of MARCKS subcellular distribution, mice from each strain were subjected to 60 min restraint stress and MARCKS subcellular distribution was determined 24 h later. Restraint stress resulted in a significant reduction in membrane MARCKS expression in C57BL/6J hippocampus but not in the DBA/2J hippocampus despite similar stress‐induced increases in serum corticosterone. Restraint stress did not affect cytosolic or total MARCKS levels in either strain. Similarly, restraint stress (30 min) in rats also induced a significant reduction in membrane MARCKS, but not total or cytosolic MARCKS, in the hippocampus but not in frontal cortex. In rats, chronic lithium treatment prior to stress exposure reduced hippocampal MARCKS expression but did not affect the stress‐induced reduction in membrane MARCKS. Collectively these data demonstrate higher resting levels of MARCKS in the hippocampus of DBA/2J mice compared to C57BL/6J mice, and that acute stress leads to a long‐term reduction in membrane MARCKS expression in C57BL/6J mice and rats but not in DBA/2J mice. These strain differences in hippocampal MARCKS expression and subcellular translocation following stress may contribute to the differences in behaviors requiring hippocampal plasticity observed between these strains.

The clinical efficacy of lithium in the prophylaxis of recurrent affective episodes in bipolar disorder is characterized by a lag in onset and remains for weeks to months after discontinuation. Thus, the long-term therapeutic effect of lithium likely requires reprogramming of gene expression. Protein kinase C and glycogen synthase kinase-3 signal transduction pathways are perturbed by chronic lithium at therapeutically relevant concentrations and have been implicated in modulating synaptic function in nerve terminals. These signaling pathways offer an opportunity to model critical signals for altering gene expression programs that underlie adaptive responses of neurons to long-term lithium exposure. While the precise physiological events critical for the clinical efficacy of lithium remain unknown, we propose that linking lithium-responsive genes as a regulatory network will provide a strategy to identify signature gene expression patterns that distinguish between therapeutic and nontherapeutic actions of lithium.

The search for genes in bipolar disorder has provided numerous genetic loci that have been linked to susceptibility to developing the disorder. However, because of the genetic heterogeneity inherent in bipolar disorder, additional strategies may need to be employed to fully dissect the genetic underpinnings. One such strategy involves reducing complex behaviors into their component parts (endophenotypes). Abnormal neurophysiological, biochemical, endocrinological, neuroanatomical, cognitive, and neuropsychological findings are characteristics that often accompany psychiatric illness. It is possible that some of these may eventually be useful in subdefining complex genetic disorders, allowing for improvements in diagnostic assessment, genetic linkage studies, and development of animal models. Findings in patients with bipolar disorder that may eventually be useful as endophenotypes include abnormal regulation of circadian rhythms (the sleep/wake cycle, hormonal rhythms, etc.), response to sleep deprivation, P300 event-related potentials, behavioral responses to psychostimulants and other medications, response to cholinergics, increase in white matter hyperintensities (WHIs), and biochemical observations in peripheral mononuclear cells. Targeting circadian rhythm abnormalities may be a particularly useful strategy because circadian cycles appear to be an inherent evolutionarily conserved function in all organisms and have been implicated in the pathophysiology of bipolar disorder. Furthermore, lithium has been shown to regulate circadian cycles in diverse species, including humans, possibly through inhibition of glycogen synthase kinase 3-beta (GSK-3beta), a known target of lithium.

People with schizophrenia exhibit impaired ability to modify electroencephalographic event-related potential (ERP) responses to novel stimuli. These deficits serve as a window into the abnormalities of neuronal organization and function and are thought to reflect a component of genetic vulnerability for schizophrenia. We describe differences among inbred mouse strains for ERPs following a novelty detection paradigm, as a model for genetic contributions to disease vulnerability. Auditory-evoked potentials were recorded during an auditory oddball task in nonanesthetized C57BL/6J, C3H/HeJ, and DBA/2J mice prior to and following ketamine (10 mg/kg). Stimuli consisted of 80 sets of 24 standard tones followed by one novel tone. Principal component analysis yielded four temporal components that contribute to the auditory ERP responses to standard and novel stimuli. Two principal components that varied between standard and novel stimuli also differed among inbred mouse strains. Post hoc analyses indicate that strain effects on novelty detection are due to a significant difference between the response to novel and standard tones in C3H/HeJ mice that is absent in the other two strains. Inbred strains of mice vary in their ability to perform neuronal detection of change in the auditory environment. The ability to model novelty detection deficits in mice will aid in identifying genetic contributions to abnormal neuronal organization in people with schizophrenia.

We have shown that exposure of rats to neonatal handling/maternal separation results in mossy fiber axon hypoplasia in field CA3 of the hippocampus. To better understand the molecular basis of this neuroanatomical alteration, the present study examined three developmentally regulated protein kinase C substrate mRNAs that are highly expressed in hippocampal granule cells during mossy fiber outgrowth: GAP-43, a presynaptic substrate implicated in axonal outgrowth, RC3 (neurogranin), a postsynaptic substrate implicated in calmodulin signaling, and MARCKS-like protein (MLP), which binds calmodulin and filamentous actin in neurons and glial cells. mRNA expression was examined by quantitative in situ hybridization in the developing [postnatal day 7 (P7), P13, P21, and P90] hippocampus (CA1, CA3, granule cells) in Long-Evans hooded rats: (1) reared under normal animal facility (AFR) conditions, (2) subjected to brief (15 min/day, HMS15), or (3) subjected to moderate (180 min/day) handling/maternal separation (HMS180) on P2–14. RC3 mRNA expression was consistently elevated in all of the hippocampal cell fields in HMS180 rats relative to HMS15 and/or AFR rats over postnatal development, but did not differ from HMS15 rats in adulthood. In contrast, neither GAP-43 mRNA nor MLP mRNA expression differed among AFR, HMS15, or HMS180 rats at any postnatal time point. Elevations in RC3 expression would be predicted to perturb calcium-calmodulin signaling that may, in turn, impair the formation and/or maintenance of mossy fiber-CA3 synapses during postnatal development.

Mouse MARCKS is a prominent myristoylated alanine-rich C kinase substrate implicated in brain development, calcium/calmodulin signaling, and membrane cytoskeletal restructuring, and is developmentally regulated in a cell- and tissue-specific fashion. In this study, transcriptional regulation of mouse MARCKS promoter in the neuronally derived immortalized hippocampal cells (HN33) was examined for a portion of 5′-flanking genomic sequence from −993 to +1 relative to the translation start site. Transfection experiments carried out in this neural cell line identified, for the first time, that the distal promoter segment from −993 to −713 plays a crucial role as an enhancer/activator element in the up-regulation of the basal transcription activity driven by MARCKS core promoter sequence. Motif analyses revealed at least 12 overlapping potential transcription factor binding sites in this region, among which a prominent GA-rich sequence centered at −765 has been shown to be functionally important in the binding of Sp1 protein-like complex. Deletion of the GA-rich segment significantly reduced the MARCKS promoter activity. Further, competitive EMSA indicated two additional sites within the −993/−713 that may also interact with Sp1 protein, demonstrating that the activator function of −993/−713 is under control of multiple Sp1 transcription factors. Unlike the distal promoter sequence, the proximal core promoter sequence (−649/−438) contains a GC-rich box and a Z-DNA-forming segment and is critical to basal transcription. The deletion of −649/−438 segment has been shown to drastically impair the promoter activity even in the presence of −993/−713, suggesting that its presence is also important to the function of −993/−713. These data emphasize that the synergistic interaction between distal and proximal promoter sequences is indispensable for the optimal MARCKS promoter function in the immortalized hippocampal cells. The discovery of the activator function of the MARCKS distal promoter region, and its potential interaction with multiple Sp proteins may provide a new clue to the understanding of Macs transcriptional regulation in brain.

Bipolar disorder (BPD), the province of mood stabilizers, has long been considered a recurrent disorder. For more than 50 years, lithium, the prototypal mood stabilizer, has been known to be effective not only in acute mania but also in the prophylaxis of recurrent episodes of mania and depression. By contrast, the preponderance of past research in depression has focused on the major depressive episode and its acute treatment. It is only relatively recently that investigators have begun to address the recurrent nature of unipolar disorder (UPD) and the prophylactic use of longterm antidepressant treatment. Thus, it is timely that we address in a single chapter the most promising research relevant to the pharmacodynamics of both mood stabilizers and antidepressants. As we have outlined in Fig. 79.1, it is possible to characterize both the course and treatment of bipolar and unipolar disorder in a similar manner. Effective treatments exist for the acute phases of both disorders; maintaining both types of patients on such drugs on a long-term basis decreases the likelihood and intensity of recurrences. Further, because the drugs are given long-term, they produce a cascade of pharmacologic effects over time that are ‘‘triggered’’ by their acute effects. Both classes of psychotropic drugs incur a lag period for therapeutic onset of action, even in the acute phase; therefore, studies during the past two decades have focused on the delayed (subchronic) temporal effects of these drugs over days and weeks. Consequently, it is widely thought that the delayed pharmacologic effects of these drugs are relevant for either the initiation of behavioral improvement or the progression of improvement beyond that initiated by acute pharmacologic actions. The early realization that lithium is effective prophylactically in BPD and

Non-adherence with medication remains a major correctable cause for poor outcome in schizophrenia. We describe a surgically implantable preparation of haloperidol with the aim that patients will have superior outcomes with improved medication adherence from implants. In contrast to depot formulations, implantable pellets could last many months, providing symptomatic improvement for periods of time never before possible. Additionally, in the event of unacceptable side effects, implants could be removed, offering a degree of reversibility not available with depot formulations. A surgically-implantable formulation of haloperidol has been created using biodegradable polymers. Implants have been characterized for in-vitro kinetics, as well as in-vivo bioactivity in rodents. Haloperidol implants demonstrate steady release of drug for 5 months. Animals treated with haloperidol implants display increased striatal D2 receptor expression as well as increased apomorphine stimulated locomotion. Surgically-implantable formulations are a viable approach to provide long-term delivery of antipsychotic medications to patients with psychotic disorders.

Valproic acid (VPA) is a broad-spectrum anticonvulsant with well-documented teratogenic effects, but whose mechanism of action is largely unknown. In the present study we have examined the effects of VPA on the expression of two prominent substrates for protein kinase C (PKC) in the brain, MARCKS and GAP-43, which have been implicated in actin-membrane plasticity and neurite outgrowth during neuronal differentiation, respectively, and are essential to normal brain development. Immortalized hippocampal HN33 cells exposed to VPA exhibited reduced MARCKS protein expression and demonstrated increased GAP-43 protein expression, with concomitant alterations in cellular morphology, including an increase in the number and length of neurites and accompanied by a reduction in cell growth rate. The effects of VPA were observed at clinically relevant concentrations following chronic (>1 day) VPA exposure. We also present evidence for a VPA-induced alteration in PKC activity, as well as temporal changes in individual PKC isozyme expression. Inhibition of PKC with the PKC-selective inhibitor, LY333531, prevented the VPA-induced down-regulation of membrane-associated MARCKS, but had no effect on the cytosolic MARCKS reduction or the GAP-43 up-regulation. Inhibition of PKC by LY333531 enhanced the differentiating effects of VPA; additionally, LY333531 alone induced greater neurite outgrowth in this cell line. Collectively, these data indicate that VPA induces neuronal differentiation, associated with a reduction in MARCKS expression and an increase in GAP-43 expression, consistent with the hypothesis that a reduction in MARCKS at the membrane may be permissive for cytoskeletal plasticity during neurite outgrowth.