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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., ((c) 2006 Wiley-Liss Inc.)

The birth and differentiation of neurons have been extensively studied in the olfactory epithelium (OE) of rodents but not in humans. The goal of this study was to characterize cellular composition and molecular expression of human OE in vivo and in vitro. In rodent OE, there are horizontal basal cells and globose basal cells that are morphologically and functionally distinct. In human OE, however, there appears to be no morphological distinction among basal cells, with almost all cells having round cell bodies similar to rodent globose basal cells. Unlike the case in rodents, human basal cells, including putative neuronal precursors, express p75NGFR, suggesting a distinctive role for p75NGFR in human OE neurogenesis. Molecular expression of neuronal cells during differentiation in human OE grossly follows that in rodents. However, the topographical organization of immature and mature ORNs in human OE differs from that of rodents, in that immature and mature ORNs in humans are dispersed throughout the OE, whereas rodent counterparts have a highly laminar organization. These observations together suggest that the birth and differentiation of neuronal cells in human OE differ from those in rodents. In OE explant culture, neuronal cells derived from human OE biopsy express markers for immature and mature neurons, grossly recapitulating neuronal differentiation of olfactory neurons in vivo. Furthermore, small numbers of cells are doubly label for bromodeoxyuridine and olfactory marker protein, indicating that neuronal cells born in vitro reach maturity. These data highlight species-related differences in OE development and demonstrate the utility of explant culture for experimental studies of human neuronal development.

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., (Copyright 2005 Wiley-Liss, Inc.)

Protein kinase C gamma (PKC gamma) 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 PKC gamma 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 PKC gamma expression in the hippocampus of C57BL/6J mice compared to no-stress controls, but did not alter either membrane or cytosolic PKC gamma expression in the hippocampus of DBA/2J mice compared to no-stress controls. These data provide direct evidence that PKC gamma is differentially regulated in the hippocampus of C57BL/6J and DBA/2J mice by acute stress. The role of stress-induced regulation of hippocampal PKC gamma expression in hippocampal synaptic plasticity is discussed.

Objective: To examine whether lamotrigine has a unique role in the treatment of bipolar disorder, we evaluated the results of recent clinical trials and molecular and cell biological studies on lamotrigine., Data Sources: Using keywords such as bipolar disorder, lamotrigine, clinical trial, outcomes studies, and mechanisms, we conducted a search for English-language articles on MEDLINE and Index Medicus and also on abstracts presented in recent research conferences., Data Synthesis: Several studies have strongly suggested that lamotrigine is effective for the acute treatment of bipolar depression as well as for long-term maintenance treatment of bipolar disorder. Stevens-Johnson syndrome is a concern, but the incidence of this side effect may not be as high as previously believed, if dosing is slowly titrated. The action mechanisms underlying the mood-stabilizing effects of lamotrigine are unknown at present but recent studies have produced interesting leads. Lamotrigine modulates various ion channels, altering neuronal excitability. The use-dependent inhibition of neuronal firing by lamotrigine is potentially important because it could result in attenuating supranormal neuronal activities that are possibly associated with bipolar disorder. Lamotrigine inhibits the release of glutamate, similarly to lithium, and its possible association with mood-stabilizing or antidepressant effects needs to be further examined. Unlike lithium or valproic acid, however, lamotrigine does not down-regulate the expression of protein kinase C or MARCKS, suggesting that lamotrigine employs different intracellular mechanisms for long-term changes in neuro-biology from those of lithium or valproic acid., Conclusion: The efficacy of lamotrigine for bipolar depression may provide us with new options in the treatment of bipolar disorder. Examining the effects of lamotrigine on various molecular mechanisms in correlation with its unique efficacy on bipolar depression may enhance our understanding of action mechanisms of the mood stabilizers.

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.

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 ( approximately 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.

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.

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.

Neonatal maternal separation of rat pups leads to a stable stress hyper-responsive phenotype characterized by increased basal levels of corticotropin releasing factor (CRF) mRNA in the hypothalamic and extra-hypothalamic nuclei, increased hypothalamic CRF release, and enhanced adrenocorticotrophin hormone (ACTH) and corticosterone (CORT) responses to psychological stressors. Stress and exposure to glucocorticoids either early in life or in adulthood have been associated with hippocampal atrophy and impairments in learning and memory. In this study, male Long Evans rat pups were exposed to daily 3-h (HMS180) or 15-min (HMS15) periods of maternal separation on postnatal days (PND) 2-14 or normal animal facility rearing. Maternal separation and subsequent reunion with the dam resulted in elevated plasma CORT levels versus HMS15 animals at PND7, a time when rat pups are normally hyporesponsive to stressors and show limited pituitary-adrenal responses. As adults, HMS180 rats exhibited elevated indices of anxiety, startle-induced pituitary-adrenal hyper-responsiveness, and slight, but significant impairment on acquisition in the Morris water maze task. In addition, HMS180 rats exhibited decreased mossy fiber density in the stratum oriens region of the hippocampus as measured by Timm's staining, but no change in volume of the dentate gyrus. These changes may be the result of neonatal exposure to elevated glucocorticoids and/or changes in other signaling systems in response to maternal separation. Overall the results suggest that repeated, daily, 3-h maternal separations during critical periods of hippocampal development can disrupt hippocampal cytoarchitecture in a stable manner. The resulting change in morphology may contribute to the subtle, but consistent learning deficit and overall stress hyper-responsive phenotype observed in these animals.

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.

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., (Published 2002 Wiley-Liss, Inc.)

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.

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., ((c)2002 Elsevier Science (USA).)

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., (Copyright 2002 S. Karger AG, Basel)

The gene (Macs) for the mouse myristoylated alanine-rich C kinase substrate (MARCKS) encodes a prominent substrate for protein kinase C that has been implicated in processes requiring signal dependent changes in actin-membrane plasticity and cytoskeletal restructuring. We have previously demonstrated that MARCKS protein is significantly down-regulated in rat hippocampus and in an immortalized hippocampal cell line (HN33.dw) following long-term exposure to lithium at clinically relevant concentrations (1 mM). Our current studies have examined transcriptional and post-transcriptional events that may underlie the lithium-induced down-regulation of MARCKS protein in the cultured hippocampal cell model system. MARCKS mRNA and protein expression were found to be concomitantly down-regulated following exposure of the HN33.dw cells to chronic lithium. Whereas the stability of MARCKS mRNA remained unchanged in the presence of lithium, nuclear run-off assay indicated that the transcription of nascent MARCKS mRNA was significantly reduced (approximately 50%) in the cells that had been treated with lithium for 7 days. Transient transfection of HN33.dw cells with a mouse cloned Macs promoter (993-bp) showed that the Macs promoter activity was attenuated to the same extent after chronic (7-10 days), but not subacute (24 h), lithium exposure. The inhibition of the Macs promoter was found to be dependent upon the presence of a 280-bp promoter region between -993-bp and -713-bp relative to the translation start site, suggesting that this region is a potential lithium-responsive region of Macs promoter (LRR). Mutant promoter lacking the LRR not only did not respond to chronic lithium exposure but also had significantly reduced promoter activity, suggesting that chronic lithium exposure represses the transcriptional activity of activator(s) bound to the promoter. Taken together, our data indicate that transcriptional inhibition of the Macs gene underlies the lithium-induced down-regulation of MARCKS expression in the immortalized hippocampal cells.

We recently identified conformational changes that occur upon phosphorylation of myristoylated alanine-rich protein kinase C substrate (MARCKS) that preclude efficient cross-linking of actin filaments (Bubb, M. R., Lenox, R. H., and Edison, A. S. (1999) J. Biol. Chem. 274, 36472-36478). These results implied that the phosphorylation site domain of MARCKS has two actin-binding sites. We now present evidence for the existence of two actin-binding sites that not only mutually compete but also specifically compete with the actin-binding proteins thymosin beta(4) and actobindin to bind to actin. The effects of substitution of alanine for phenylalanine within a repeated hexapeptide segment suggest that the noncharged region of the domain contributes to binding affinity, but the binding affinity of peptides corresponding to each binding site has a steep dependence on salt concentration, consistent with presumed electrostatic interactions between these polycationic peptides and the polyanionic N terminus of actin. Phosphorylation decreases the site-specific affinity by no more than 0.7 kcal/mol, which is less than the effect of alanine substitution. However, phosphorylation has a much greater effect than alanine substitution on the loss of actin filament cross-linking activity. These results are consistent with the hypothesis that the compact structure resulting from conformational changes due to phosphorylation, in addition to modest decreases in site-specific affinity, explains the loss of cross-linking activity in phosphorylated MARCKS.

In the mature hippocampus, kainic acid seizures lead to excitotoxic cell death and synaptic reorganization in which granule cell axons (mossy fibers) form ectopic synapses on granule cell dendrites. In the present study, we examined the expression of four major, developmentally regulated protein kinase C (PKC) substrates (MARCKS, MLP, GAP-43, RC3), which have different subcellular and regional localizations in the hippocampus at several time points (6 hr, 12 hr, 18 hr, 24 hr, 48 hr, 5 days, or 15 days) following kainic acid seizures using in situ hybridization. Consistent with previous reports, following kainate seizures, GAP-43 mRNA expression exhibited a delayed and protracted elevation in the granule cell layer, which peaked at 24 hr, whereas expression in fields CA1 and CA3 remained relatively unchanged. Conversely, RC3 mRNA expression exhibited a delayed reduction in the granule cell layer that was maximal at 18 hr, as well as a reduction CA1 at 48 hr, whereas CA3 levels did not change. MARCKS mRNA expression in the granule cell layer and CA1 remained stable following kainate, although an elevation was observed in subfield CA3c at 12 hr. Similarly, MLP mRNA expression did not change in the granule cell layer or CA1 following kainate but exhibited a protracted elevation in subfields CA3b,c beginning at 6 hr post-kainate. Collectively these data demonstrate that different PKC substrate mRNAs exhibit unique expression profiles and regulation in the different cell fields of the mature hippocampus following kainic acid seizures and during subsequent synaptic reorganization. The expression profiles following kainate seizures bear resemblance to those observed during postnatal hippocampal development, which may indicate the recruitment of common regulatory mechanisms., (Copyright 2000 Wiley-Liss, Inc.)

The myristoylated alanine-rich C kinase substrate (MARCKS) is a prominent PKC-substrate in the brain, which has been implicated in brain development, cytoskeletal remodeling, calcium/calmodulin signaling, and neuroplasticity. The sequence of the Macs gene codes for a protein that has three highly conserved domains including a 5' myristoylation region and a 25-amino-acid phosphorylation site domain (PSD), which are involved in anchoring MARCKS to the cellular membrane. In this study, we examined the role of the myristoylation signal in the regulation of MARCKS in transfected rat hippocampal cells (H19-7) following retinoic acid (RA) treatment. A mutant MARCKS lacking the myristoylation signal was engineered by substitution of alanine for glycine at position 2 of the Macs gene and was found to be exclusively expressed in the cytosol fraction of transfected cells. Exposure of the wild-type MARCKS-transfected cells to RA resulted in an apparent shift of MARCKS from the membrane to the cytosol, while the total protein of wild-type MARCKS was not significantly changed. In contrast, RA-exposed cells transfected with the mutant MARCKS revealed a dramatic reduction of expression of MARCKS protein in both cytosol and total protein fractions. These data suggest that the absence of the myristoyl moiety may not only alter the anchoring of the protein to the membrane but also play a novel role in modulating cellular levels of MARCKS protein in response to RA., (Copyright 2000 Academic Press.)

Clinical studies over the years have provided evidence that monoamine signaling and hypothalamic-pituitary-adrenal axis disruption are integral to the pathophysiology of bipolar disorder. A full understanding of the pathophysiology from a molecular to a systems level must await the identification of the susceptibility and protective genes driving the underlying neurobiology of bipolar disorder. Furthermore, the complexity of the unique biology of this affective disorder, which includes the predisposition to episodic and often progressive mood disturbance, and the dynamic nature of compensatory processes in the brain, coupled with limitations in experimental design, have hindered our progress to date. Imaging studies in patient populations have provided evidence of a role for anterior cingulate, amygdala, and prefrontal cortex in the pathophysiology of bipolar disorder. More recent research strategies designed to uncover the molecular mechanisms underlying our pharmacologic treatments and their interaction in the regulation of signal transduction as well as more advanced brain imaging studies remain promising approaches. This experimental strategy provides data derived from the physiologic response of the system in affected individuals and addresses the critical dynamic interaction with pharmacologic agents that effectively modify the clinical expression of the pathophysiology.

Since its discovery, lithium has been shown to act upon various neurotransmitter systems at multiple levels of signaling in the brain. Lithium, affecting each neurotransmitter system within complex interactive neuronal networks, is suggested to restore the balance among aberrant signaling pathways in critical regions of the brain. Recent molecular studies have revealed the action of lithium on signal transduction mechanisms, such as phosphoinositide hydrolysis, adenylyl cyclase, G protein, glycogen synthase kinase-3beta, protein kinase C, and its substrate myristoylated alanine-rich C kinase substrate. Such effects are thought to trigger long-term changes in neuronal signaling patterns that account for the prophylactic properties of lithium in the treatment of bipolar disorder. Through its effects on glycogen synthase kinase-3beta and protein kinase C, lithium may alter the level of phosphorylation of cytoskeletal proteins, which leads to neuroplastic changes associated with mood stabilization. Chronic lithium regulates transcriptional factors, which in turn may modulate the expression of a variety of genes that compensate for aberrant signaling associated with the pathophysiology of bipolar disorder. Future studies on long-term neuroplastic changes caused by lithium in the brain will set the stage for new drug-discovery opportunities.

We have previously shown that the myristoylated alanine-rich C kinase substrate, a primary protein kinase C substrate in brain that binds and cross-links filamentous actin, is enriched in neuronal growth cones and is developmentally regulated in brain. Here we examined myristoylated alanine-rich C kinase substrate expression in the facial motor nucleus during axonal regeneration following facial nerve axotomy or facial nerve resection lesions, which impede regeneration, or following motor neuron degeneration induced by the retrograde neurotoxin ricin. For comparative purposes, the protein kinase C substrates myristoylated alanine-rich C kinase substrate-like protein and growth-associated protein-43 were examined in parallel. Myristoylated alanine-rich C kinase substrate messenger RNA exhibited a robust increase in both neurons and non-neuronal cells in the facial motor nucleus beginning four days after axotomy, peaked at seven days (2.5-fold), and declined back to baseline levels by 40 days. Myristoylated alanine-rich C kinase substrate protein similarly exhibited a twofold elevation in the facial motor nucleus determined four and 14 days post-axotomy. Following nerve resection, myristoylated alanine-rich C kinase substrate messenger RNA levels increased at seven days and returned to baseline levels by 40 days. Unlike myristoylated alanine-rich C kinase substrate messenger RNA, myristoylated alanine-rich C kinase substrate-like messenger RNA levels did not increase in the facial motor nucleus at any time point following nerve axotomy or resection, whereas growth-associated protein-43 messenger RNA exhibited a rapid (one day) and prolonged (40 days) elevation in facial motor nucleus neurons following either nerve axotomy or resection. Ricin-induced degeneration of facial motor neurons elevated myristoylated alanine-rich C kinase substrate and myristoylated alanine-rich C kinase substrate-like messenger RNAs in both microglia (lectin-positive) and astrocytes (glial fibrillary acidic protein-positive).Collectively, these data demonstrate that myristoylated alanine-rich C kinase substrate exhibits a unique expression profile in the facial motor nucleus following facial nerve lesions, and it is proposed that myristoylated alanine-rich C kinase substrate may serve to mediate actin-membrane cytoskeletal plasticity in both neurons and glial cells in response to protein kinaseC-mediated signaling during nerve regeneration and degeneration.

The underlying pathophysiology of bipolar disorder is a continually evolving complexity of multilayer interacting and independent systems. The dearth of adequate preclinical or clinical models that incorporate the various features of the illness, i.e., acute and chronic, recurrent and episodic, and time-course and treatment-related variables, has made the consistency and interpretation of data difficult. Newer technologies and the availability of structurally and mechanistically distinct pharmacologic agents have expanded opportunities for experimental study. In addition to the well-known neurotransmitter systems that are disrupted in mood disorders, critical guanine nucleotide-binding protein (G protein)-coupled signaling pathways are implicated in modulating mood state. Regulation of gene expression and identification of factors regulating neuroplasticity and neurotrophic events in the central nervous system in bipolar disorder are 2 of the more recent approaches contributing to clarification of the pathophysiology and potential treatment options.

Phosphorylation of myristoylated alanine-rich protein kinase C substrate (MARCKS) by protein kinase C eliminates actin filament cross-linking activity, but residual filament binding activity docks phosphorylated MARCKS on filamentous actin. The postulated actin-binding region of MARCKS, which includes a Ca(2+)-calmodulin-binding site, has been portrayed with alpha-helical structure, analogous to other calmodulin-binding domains. Previous speculation suggested that MARCKS may dimerize to form the two functional actin-binding sites requisite for cross-linking activity. Contrary to these hypotheses, we show that MARCKS peptide with actin-cross-linking activity has an extended structure in aqueous solution but assumes a more compact structure upon phosphorylation. We hypothesize that structural changes in the MARCKS peptide induced by phosphorylation create a dynamic structure that, on average, has only one actin-binding site. Moreover, independent of the state of phosphorylation, this peptide is monomeric rather than dimeric, implying that two distinct actin-binding sites are responsible for the actin-cross-linking activity of unphosphorylated MARCKS. These studies uniquely elucidate the mechanism by which phosphorylation of MARCKS induces structural changes and suggest how these structural changes determine biological activity.

The long-term treatment of manic-depressive illness (MDI) likely involves the strategic regulation of signalling pathways and gene expression in critical neuronal circuits. Accumulated evidence has identified signalling pathways, in particular the family of protein kinase C (PKC) isozymes, as targets for the long-term action of lithium. Chronic lithium administration produces a reduction in the expression of PKC alpha and epsilon, as well as a major PKC substrate, MARCKS, which has been implicated in long-term neuroplastic events in the developing and adult brain. More recently, studies have demonstrated robust effects of lithium on another kinase system, GSK-3beta, and on neuroprotective/neurotrophic proteins in the brain. Given the key roles of these signalling cascades in the amplification and integration of signals in the central nervous system, these findings have clear implications not only for research into the neurobiology of MDI, but also for the future development of novel and innovative treatment strategies.

Understanding the biology of the pharmacological stabilization of mood will undoubtedly serve to provide significant insight into the pathophysiology of manic-depressive illness (MDI). Accumulating evidence from our laboratories and those of other researchers has identified the family of protein kinase C isozymes as a shared target in the brain for the long-term action of both lithium and valproate. In rats chronically treated with lithium, there is a reduction in the hippocampus of the expression of two protein kinase isozymes, alpha and epsilon, as well as a reduction in the expression of a major PKC substrate, MARCKS, which has been implicated in long-term neuroplastic events in the developing and adult brain. In addition, we have been investigating the down-stream impact of these mood stabilizers on another kinase system, GSK-3 beta and on the AP-1 family of transcription factors. Further studies have generated promising preliminary data in support of the antimanic action of tamoxifen, and antiestrogen that is also a PKC inhibitor. Future studies must address the therapeutic relevance of these protein targets in the brain using innovative strategies in both animal and clinical investigations to ultimately create opportunities for the discovery of the next generations of mood stabilizers for the treatment of MDI.

Activation of protein kinase C (PKC) is one of the biochemical pathways thought to be activated during activity-dependent synaptic plasticity in the brain, and long-term potentiation (LTP) and long-term depression (LTD) are two of the most extensively studied models of synaptic plasticity. Here we have examined changes in the in situ phosphorylation level of two major PKC substrates, myristoylated alanine-rich C kinase substrate (MARCKS) and growth-associated protein (GAP)-43/B-50, after pharmacological stimulation or induction of LTP or LTD in the CA1 field of the hippocampus. We find that direct PKC activation with phorbol esters, K+-induced depolarization, and activation of metabotropic glutamate receptors increase the in situ phosphorylation of both MARCKS and GAP-43/B-50. The induction of LTP increased the in situ phosphorylation of both MARCKS and GAP-43/B-50 at 10 min following high-frequency stimulation, but only GAP-43/B-50 phosphorylation remained elevated 60 min after LTP induction. Furthermore, blockade of LTP induction with the NMDA receptor antagonist D-2-amino-5-phosphonopentanoic acid prevented elevations in GAP-43/B-50 phosphorylation but did not prevent the elevation in MARCKS phosphorylation 10 min following LTP induction. The induction of LTD resulted in a reduction in GAP-43/B-50 phosphorylation but did not affect MARCKS phosphorylation. Together these findings show that activity-dependent synaptic plasticity elicits PKC-mediated phosphorylation of substrate proteins in a highly selective and coordinated manner and demonstrate the compartmentalization of PKC-substrate interactions. Key Words: Protein kinase C-Myristoylated alanine-rich C kinase substrate-Growth-associated protein-43-Long-term potentiation-Long-term depression-(RS)-alpha-Methyl-4-carboxyphenylglycine-D-2-Amino-5-ph osphonopentanoic acid-Glutamate.

The expression of GAP-43 was modulated genetically in the adult rat nigrostriatal or septohippocampal pathway using recombinant adeno-associated virus (rAAV) vectors incorporating the neuron specific enolase (NSE) promoter and either a rat GAP-43 cDNA or the corresponding antisense sequence. Bicistronic expression of green fluorescent protein (GFP) enabled us to evaluate transduced neurons selectively. Single injections of rAAV into the substantia nigra pars compacta (SNc) transduced both dopaminergic and non-dopaminergic neurons stably for the 3-month duration of the study. Transduction with the GAP-43 vector in this region: (1) increased GAP-43 mRNA levels 2-fold compared to controls; (2) led to GAP-43 immunoreactivity in neuronal perikarya, axons, and dendrites that was not observed otherwise; and (3) resulted in GAP-43/ GFP-positive axons that were traced to the striatum where they formed clusters of aberrant nets. The GAP-43 antisense vector, in contrast, decreased neuropil GAP-43 immunoreactivity compared to controls in the SNc. In septum, injections of the GAP-43 expressing vector also caused aberrant clusters of GAP-43 labelled fibers in terminal fields, i.e., fornix and hippocampus, that were not observed in control tissues. It therefore appears that rAAV vectors provide a novel approach for modulating intraneuronal GAP-43 expression in the adult brain., (Copyright 1999 Elsevier Science B.V.)

Protein kinase C (PKC) consists of a family of Ca2+/phospholipid-dependent isozymes that has been implicated in the delayed neurotoxic effects of glutamate in vitro. In the present study, we assessed the effect of the glutamate analogue kainic acid (KA) on the subcellular expression of PKC isozymes in the hippocampus (HPC) in the period preceding (0.5, 1.5, 12, and 24 h) and during (120 h) hippocampal necrosis using western blot analysis and PKC isozyme-specific antibodies. Before subcellular fractionation (cytosol + membrane), hippocampi were microdissected into "HPC" (fields CA1-CA3) and "dentate gyrus" (DG; granule cells + hilus) regions. Four general patterns of alterations in PKC isozyme expression/distribution were observed following KA treatment. The first pattern was a relative stability in expression following KA treatment and was most apparent for cytosol PKCalpha (HPC + DG) and membrane (HPC) and cytosol (DG) PKCbetaII. The second pattern, observed with PKCgamma and PKCepsilon, was characterized by an initial increase in expression in both membrane and cytosolic fractions before seizure activity (0.5 h) followed by a gradual decrease until significant reductions are observed by 120 h. The third pattern, exhibited by PKCdelta, involved an apparent translocation, increasing in the membrane and decreasing in the cytosol, followed by down-regulation in both fractions and subsequent recovery. The fourth pattern was observed with PKCzeta only and entailed a significant reduction in expression before and during limbic motor seizures followed by a dramatic fivefold increase in the membrane fraction during the period of hippocampal necrosis (120 h). Although these patterns did not segregate according to conventional PKC isozyme classifications, they do indicate dynamic isozyme-specific regulation by KA. The subcellular redistribution of PKC isozymes may contribute to the histopathological sequelae produced by KA in the hippocampus and may model the pathogenesis associated with diseases involving glutamate-induced neurotoxicity.

Background: Although suicide is a leading cause of death in the United States and represents a significant public health threat, little is known about the neurobiological or molecular factors that contribute to its pathophysiology. A number of studies now indicate that lithium has considerable efficacy in the prevention of suicide in patients with affective disorders, and accumulating evidence indicates that protein kinase C (PKC) and its substrates, in particular the myristoylated alanine-rich C kinase substrate (MARCKS), are primary targets of chronic lithium treatment. We therefore hypothesized that a dysregulation in MARCKS expression in key brain regions could contribute to the pathophysiology associated with suicide. To address this, we examined MARCKS, as well as the closely related MARCKS-related protein (MRP), mRNA expression in the hippocampus and dorsolateral prefrontal cortex of suicide victims and normal controls., Method: MARCKS and MRP mRNA expression was assessed by quantitative in situ hybridization histochemistry performed on postmortem hippocampal and dorsolateral prefrontal cortex sections from suicide (N = 9) and normal control (N = 10) brains., Results: In the normal hippocampus, both MARCKS and MRP mRNA expression were highest in the granule cell layer and low-moderate in CA1, CA3, and hilus. A high level of MRP mRNA expression was also observed in the white matter of the fimbria/fornix. Neither MARCKS nor MRP mRNA expression levels differed significantly in the granule cell layer, CA3, hilus, or CA1 in suicide victims relative to normal controls (1-way ANOVA, p > .05). In the normal prefrontal cortex, MARCKS was expressed exclusively in gray matter (layers I-VI), whereas MRP was expressed in both gray and white matter. Neither MARCKS nor MRP mRNA expression levels in the gray and white matter regions of the dorsal prefrontal cortex differed between suicides and normal controls (1-way ANOVA, p > .05)., Conclusion: The present findings are the first to demonstrate the expression and distribution of MARCKS and MRP in the human hippocampus and dorsolateral prefrontal cortex, and their expression pattern within these regions bears strong resemblance to those observed in the adult rat brain. Comparison of MARCKS and MRP mRNA expression in the hippocampus and prefrontal cortex of suicide victims and normal controls indicates that these 2 mRNAs are not differentially regulated in these regions. However, differences in MARCKS and MRP protein expression and function cannot be ruled out by the present findings.

The myristoylated alanine-rich C kinase substrate (MARCKS) is a prominent protein kinase C (PKC) substrate in brain that is expressed highly in hippocampal granule cells and their axons, the mossy fibers. Here, we examined hippocampal infrapyramidal mossy fiber (IP-MF) limb length and spatial learning in heterozygous Macs mutant mice that exhibit an approximately 50% reduction in MARCKS expression relative to wild-type controls. On a 129B6(N3) background, the Macs mutation produced IP-MF hyperplasia, a significant increase in hippocampal PKCepsilon expression, and proficient spatial learning relative to wild-type controls. However, wild-type 129B6(N3) mice exhibited phenotypic characteristics resembling inbred 129Sv mice, including IP-MF hypoplasia relative to inbred C57BL/6J mice and impaired spatial-reversal learning, suggesting a significant contribution of 129Sv background genes to wild-type and possibly mutant phenotypes. Indeed, when these mice were backcrossed with inbred C57BL/6J mice for nine generations to reduce 129Sv background genes, the Macs mutation did not effect IP-MF length or hippocampal PKCepsilon expression and impaired spatial learning relative to wild-type controls, which now showed proficient spatial learning. Moreover, in a different strain (B6SJL(N1), the Macs mutation also produced a significant impairment in spatial learning that was reversed by transgenic expression of MARCKS. Collectively, these data indicate that the heterozygous Macs mutation modifies the expression of linked 129Sv gene(s), affecting hippocampal mossy fiber development and spatial learning performance, and that MARCKS plays a significant role in spatial learning processes.

The myristoylated alanine-rich C kinase substrate (MARCKS) and MARCKS-related protein (MRP) are both membrane-associated phosphoproteins that interact with calmodulin and filamentous actin in a protein kinase C phosphorylation-dependent manner. In the present study, we examined MARCKS and MRP gene expression in the postnatal (P) rat brain (1, 7, 14, 21, and 90 days after birth) by using quantitative in situ hybridization. At P1, MRP expression was high in neocortex, striatum, thalamus, cerebellar cortex, and hippocampus (CA1-CA3, hilus, and granule cell layer) but low in brainstem and, between P7 and P14, exhibited a dramatic decline in each of these regions except hippocampal CA1 and granule cell layers. Between P14 and P21, MRP expression increased in white matter regions including the corpus callosum, fimbria/fornix, and cerebellar deep white matter. At P90 (adult), MRP remained strongly expressed in the olfactory bulb, medial habenula, hippocampal CA1, and the inner two-thirds of granule cell layer, temporal, and entorhinal cortices, the corpus callosum and fimbria/fornix, and cerebellar white matter. At P1, MARCKS was strongly expressed in the majority of brain regions except the brainstem, which subsequently declined gradually to approximate adult levels by P14. Between P14 and P21, MARCKS expression declined gradually in the hilus, remained elevated in hippocampal CA1, CA3, and granule cell layers, and increased dramatically in the corpus callosum and fimbria/fornix. At P90, MARCKS expression declined in hippocampal CA3 and hilus and remained strongly expressed in hippocampal CA1 and granule cell layers, regions of the olfactory bulb, the medial habenula, temporal cortex, and cerebellar granule and Purkinje cells. Expression of both MARCKS and MRP in regions undergoing neuronal proliferation, migration, and neurite outgrowth suggest a common role in these developmental events, whereas differences in expression during development and in the adult brain provide evidence of differential regulation.

Angiotensin II (Ang II) exerts chronic stimulatory actions on tyrosine hydroxylase (TH), dopamine beta-hydroxylase (DbetaH), and the norepinephrine transporter (NET), in part, by influencing the transcription of their genes. These neuromodulatory actions of Ang II involve Ras-Raf-MAP kinase signal transduction pathways (Lu, D., H. Yang, and M.K. Raizada. 1997. J. Cell Biol. 135:1609-1617). In this study, we present evidence to demonstrate participation of another signaling pathway in these neuronal actions of Ang II. It involves activation of protein kinase C (PKC)beta subtype and phosphorylation and redistribution of myristoylated alanine-rich C kinase substrate (MARCKS) in neurites. Ang II caused a dramatic redistribution of MARCKS from neuronal varicosities to neurites. This was accompanied by a time-dependent stimulation of its phosphorylation, that was mediated by the angiotensin type 1 receptor subtype (AT1). Incubation of neurons with PKCbeta subtype specific antisense oligonucleotide (AON) significantly attenuated both redistribution and phosphorylation of MARCKS. Furthermore, depletion of MARCKS by MARCKS-AON treatment of neurons resulted in a significant decrease in Ang II-stimulated accumulation of TH and DbetaH immunoreactivities and [3H]NE uptake activity in synaptosomes. In contrast, mRNA levels of TH, DbetaH, and NET were not influenced by MARKS-AON treatment. MARCKS pep148-165, which contains PKC phosphorylation sites, inhibited Ang II stimulation of MARCKS phosphorylation and reduced the amount of TH, DbetaH, and [3H]NE uptake in neuronal synaptosomes. These observations demonstrate that phosphorylation of MARCKS by PKCbeta and its redistribution from varicosities to neurites is important in Ang II-induced synaptic accumulation of TH, DbetaH, and NE. They suggest that a coordinated stimulation of transcription of TH, DbetaH, and NET, mediated by Ras-Raf-MAP kinase followed by their transport mediated by PKCbeta-MARCKS pathway are key in persistent stimulation of Ang II's neuromodulatory actions.

Lithium's therapeutic specificity for the treatment of bipolar disorder may be attributable in part to an ability to target sites where there are high levels of synaptic activity. We show that glutamate receptors expressed in oocytes are highly permeable to lithium. Mathematical simulations of Li+ diffusion in mature dendritic spines suggest that in the presence of 1 mM extracellular lithium one synaptic current can increase Li+ concentration in the spine head by 4 mM with a decay time constant of about 15-20 ms. Two or more current spikes will produce oscillations between 6 and 8 mM or potentially higher. These results predict that the local intracellular lithium in dendritic spines can rise to high enough levels to uncouple second messenger mechanisms of coincidence detection.

Sodium valproate (VPA) is a short-chain fatty acid with well-established anticonvulsant properties and apparent clinical efficacy in the treatment of bipolar disorder (manic-depressive illness). Little is known regarding the mechanism of action of VPA in the brain that could account for this clinical therapeutic profile. Lithium has been the standard treatment for bipolar disorder, and it is known to be an uncompetitive inhibitor of inositol monophosphatase in the phosphoinositide (PI) signaling cascade at clinically relevant concentrations. Recent studies have provided data in support of a role for protein kinase C and the down-regulation of expression of the myristoylated alanine-rich C kinase substrate (MARCKS) in the long-term therapeutic action of lithium in the brain, which is dependent on both the relative activity of receptor-coupled PI signaling and the concentration of myo-inositol. Our current results demonstrated that valproate induces a concentration- and time-dependent reduction of MARCKS in immortalized hippocampal cells that appears to be independent of both the level of muscarinic receptor-activated PI signaling as well as the concentration of myo-inositol. In CHO-K1 cells transfected with the human m1 muscarinic receptor, unlike lithium, there is no evidence for receptor-mediated accumulation of CMP-PA in the presence of VPA, providing more direct data for its lack of interaction within the PI signaling cascade. The action of VPA on MARCKS occurs within the therapeutic concentrations and time course observed in clinical studies of patients with bipolar disorder. Furthermore, the effect on MARCKS protein is additive in the presence of therapeutic concentrations of both lithium and valproate, consistent with clinical observations regarding the enhanced efficacy of the combination treatment. Finally, in studies examining acute and chronic effects of a variety of psychotropic compounds and VPA structural analogs, it is evident that the property of regulation of MARCKS is shared by the mood-stabilizers lithium and VPA, which may be specific to a class of drugs effective in the treatment of bipolar disorder.

Lithium remains a first-line approach for the treatment of acute mania and the prophylactic management of manic-depressive illness, yet the underlying neurobiological mechanisms remain as yet undefined. In this paper we critically examine the accumulated preclinical and clinical evidence for the action of lithium in the brain and suggest areas that may be most productive for future investigation, i.e., membrane transport systems, neurotransmitter receptor regulation, second messenger generating systems, protein kinase C (PKC) regulation, and gene expression. In their experimental design, preclinical investigations have often jeopardized the physiologic relevance of their studies by a relative lack of attention to issues such as therapeutic concentrations, acute versus chronic exposure, and a lack of adequate cation and/or psychotropic controls. Future studies should account for the established prophylactic efficacy of lithium, the higher risk for relapse into mania after abrupt discontinuation, the ability of lithium to stabilize recurrent depression associated with unipolar disorder, and the efficacy of lithium in the treatment of refractory major depressive disorder in the presence of an antidepressant. Studies of the action of lithium in receptor mediated phosphoinositide signaling in the brain over the past several years have opened up heuristic lines of investigation that stem from lithium's uncompetitive inhibition of the enzyme inositol monophosphatase. Subsequent studies involving regulation of inositol transport, PKC isozymes and activity, and the expression of the major PKC substrate MARCKS (myristoylated alanine-rich C-kinase substrate) have offered potential avenues for understanding the complexity of the action of long-term lithium in the brain. These studies will offer us a better understanding of the neuroanatomical sites of action of lithium and together with ongoing clinical investigations using brain imaging in patients with manic-depressive illness a more complete understanding of the pathophysiology of this disease.

The Xenopus oocyte has been shown to be a useful model for the study of signal transduction pathways. The present study investigated whether or not the oocyte could be used to study the effects of lithium on signal transduction mechanisms by comparing the dynamics of lithium homeostasis in the oocyte and a human immortalized hippocampal cell line using Flame Atomic Emission Spectroscopy (FAES). A biphasic pattern of lithium uptake was observed in the oocyte in the presence of 5 mM extracellular lithium. The late phase of lithium uptake, which started after 30 minutes of incubation time, was sensitive to phloretin, an inhibitor of Na+/Li+ counter-transport. Differences in lithium efflux kinetics further characterized the two observed phases of accumulation and also suggested that lithium might be distributed in different pools within the oocyte, including one sequestered in organelles or associated with cytosolic proteins. An analogous sequestered pool was not, however, observed in the hippocampal cell line indicating that lithium is distributed differently in these cell types. This suggests that the Xenopus oocyte might not be a suitable model for evaluating the effects of lithium on signal transduction pathways because of the unknown contribution of the sequestered pool on predicting relevant physiological effects.

Myristoylated alanine-rich C-kinase substrate (MARCKS) and F1/GAP-43 (B-50/neuromodulin) are both major specific substrates for protein kinase C (PKC) and appear to play an important role in the regulation of neuroplastic events during development and in the adult brain. Since PKC isozymes are differentially expressed in brain and the expression of F1/GAP-43 and MARCKS are differentially regulated by PKC through posttranslational mechanisms, the present study examined the relative distribution of both mRNAs in the adult brain by using in situ hybridization histochemistry. MARCKS hybridization was most pronounced in the olfactory bulb, piriform cortex (layer II), medial habenular nucleus, subregions of the amygdala, specific hypothalamic nuclei, hippocampal granule cells, neocortex, and cerebellar cortex, intermediate in the superior colliculus, hippocampal CA1, and certain brainstem nuclei including the locus coeruleus, and low-absent in regions of the caudate-putamen, geniculate, thalamic nuclei, lateral habenular nucleus, and hippocampal CA3 pyramidal and hilar neurons. Consistent with previous reports, prominent F1/GAP-43 hybridization was observed in neocortex, medial geniculate, piriform cortex (layer II), substantia nigra pars compacta, hippocampal CA3 pyramidal cells, thalamic and hypothalamic nuclei, lateral habenular nucleus, locus coeruleus, raphe nuclei, and cerebellar granule cells, intermediate in regions of the thalamus, hypothalamus, and amygdala, and low-absent in regions of the olfactory bulb, caudate-putamen, medial habenular nucleus, hippocampal granule cells, and superior colliculus. Overall, F1/GAP-43 was highly expressed in a greater number of regions compared to MARCKS and, in a number of regions, including the hippocampus, habenular complex, ventral tegmentum, geniculate, and certain brain stem nuclei, a striking inverse pattern of expression was observed. These results indicate that MARCKS gene expression, like that of F1/GAP-43, remains elevated in select regions of the adult rat brain which are associated with a high degree of retained plasticity. The potential role of PKC in the regulation of MARCKS and F1/GAP-43 gene expression in brain is assessed.

Fifty-two subjects with panic disorder and generalized anxiety disorder were interviewed 12-18 months after they completed an acute treatment study (9-11 weeks) with alprazolam followed by blind and controlled drug discontinuation. Patients were questioned about the severity of their anxiety disorder and degree of functional impairment at the time of follow-up, as well as about their subjective experience as participants in a clinical trial. At the time of follow-up 78% of the patients reported none or minimal anxiety symptoms and 89% had none or only minimal functional deficit, as compared to 100% reporting moderate to severe anxiety symptoms and 57% reporting significant functional deficit before entering the study. Overall, patients felt that the participation in the study was a very positive and beneficial experience and 64% said they would not hesitate to participate in a clinical trial in the future.

Previous studies in our laboratory have demonstrated that exposure of rats to chronic lithium results in a significant reduction in the hippocampus of levels of the protein kinase C (PKC) phosphoprotein substrate MARCKS (myristoylated alanine-rich C kinase substrate), which persists after withdrawal and is not observed following acute administration. In an immortalized hippocampal cell line (HN33), we have determined that phorbol esters rapidly down-regulate PKC activity and lead to a subsequent PKC-dependent reduction in content of MARCKS protein. We now report that chronic exposure of HN33 cells to LiCl (1-10 mM) produces a dose- and time-dependent down-regulation of MARCKS protein. The lithium-induced reduction in MARCKS is dependent on the concentration of inositol present in the medium and is reversed and prevented in the presence of elevated inositol concentrations. When HN33 cells were exposed to lithium at clinically relevant concentrations (11 mM) under limiting inositol conditions, activation of muscarinic receptor-coupled phosphoinositide signaling significantly potentiated the lithium-induced down-regulation of MARCKS protein. It has been suggested that a major action of lithium in the brain is linked to its inositol monophosphatase inhibitory activity in receptor-mediated signaling through the inositol trisphosphate/diacylglycerol pathway, resulting in a relative inositol depletion. Our data provide evidence that this initial action of lithium may translate into a PKC-dependent long-term down-regulation of MARCKS protein expression in the hippocampus.

Background: Lithium remains a first-line treatment for the acute and prophylactic management of bipolar illness. Previous studies in our laboratory have demonstrated that chronic, but not acute, exposure to therapeutic concentrations of lithium significantly reduces the expression of the protein kinase C (PKC) substrate MARCKS (myristoylated alanine-rich C kinase substrate) in the rat hippocampus and an immortalized hippocampal cell line (HN33). The anticonvulsant drugs valproate and carbamazepine are emerging as efficacious alternative and adjunctive treatments for bipolar disorder. In the present study, we sought to determine the effects of valproate and carbamazepine on MARCKS protein levels by using our hippocampal cell model., Method: HN33 immortalized hippocampal cells were exposed acutely or chronically to sodium valproate 1 mM, carbamazepine 100 microM, lithium chloride 5 mM, or lithium chloride 5 mM + sodium valproate 1 mM. Additionally, cells were exposed to lithium chloride 5 mM in the absence or presence of inositol 5 microM, or sodium valproate 1 mM in the absence or presence of inositol 40 microM. After drug exposure, cells were collected, separated into soluble and membrane fractions, and MARCKS protein assayed by Western blot analysis using polyclonal rabbit antibody. Immunoreactive bands were quantitated by densitometric analysis., Results: We report that chronic exposure of HN33 cells to either lithium or valproate produced a time-dependent down-regulation of MARCKS protein. Maximal reduction in MARCKS levels were observed after 3 days of exposure to valproate and after 7 days of exposure to lithium. The reduction of MARCKS produced by lithium and valproate alone were additive when the two drugs were combined. The reduction in MARCKS produced by lithium was reversed by the addition of inositol to the media, whereas the reduction produced by valproate was unaffected by the addition of inositol. Carbamazepine failed to affect MARCKS protein levels at each dose and time tested., Conclusion: These data provide evidence that, like lithium, chronic exposure to valproate produces a significant time-dependent down-regulation of the PKC substrate MARCKS, whereas carbamazepine is without effect. The MARCKS reduction produced by valproate appears to occur independently of inositol concentrations yet is additive with the reduction produced by lithium, which is inositol-reversible. Valproate- and lithium-induced regulation of MARCKS expression appears to be mediated by different mechanisms that may utilize PKC, and may be associated with the clinical profile of these mood stabilizers. Regulation of MARCKS expression may be associated with the prophylactic efficacy of lithium in the long-term stabilization of the recurrent affective episodes in bipolar disorder, and valproate may share this property.

Thirty-six patients with panic disorder (PD) and 35 patients with generalized anxiety disorder (GAD) participated in an open alprazolam treatment phase that preceded controlled withdrawal from alprazolam. Clinical ratings, blood pressure and heart rate were obtained along with plasma measurements of cortisol, ACTH, growth hormone and catecholamines. A similar clinical response profile was evident in both groups with rapid onset of improvement within the first week. The two diagnostic groups differed in their biological response to alprazolam. PD patients had a significant reduction in blood pressure, plasma cortisol and a trend toward significant reduction in plasma epinephrine, which were not seen in the GAD patients. GAD patients showed a significant reduction in plasma norepinephrine. These findings provide further evidence that PD and GAD are biologically distinct syndromes.

Background: An association found between akathisia and iron deficiency led to the suggestion that iron supplementation might be a useful therapeutic intervention for patients with akathisia. There is, however, a body of literature on the abnormal deposition of iron in the brain in several degenerative diseases like Hallervorden-Spatz syndrome, Parkinson's disease, and Alzheimer's disease. Given the ability of neuroleptics to chelate iron and promote its deposition in the brain, we questioned whether peripheral measures of iron are an accurate reflection of central iron levels and thus whether there was a rationale for iron supplementation in akathisia., Method: A MEDLINE search for literature relating to iron and akathisia, tardive dyskinesia, and Parkinson's disease was carried out and critically reviewed., Results: Evidence is presented for the ability of neuroleptics to chelate iron, mobilize it from peripheral stores, and deposit it in the basal ganglia. The effect of iron on dopaminergic receptor activity in brain and the potential role of iron in degenerative and neuroleptic-induced movement disorders are reviewed. The preponderance of the evidence shows a relationship between iron excess in the basal ganglia and the movement disorders. We found no studies that have examined the regulation of central levels of iron in patients with akathisia., Conclusion: The rationale for iron supplementation in the treatment of akathisia is relatively weak, and there are potentially adverse long-term consequences as outlined in our review. More research is required to directly measure the level of iron in the brain of patients with akathisia, e.g., using magnetic resonance imaging, before such therapeutic intervention can be recommended.

Lithium remains the most widely used treatment for bipolar disorder, and this monovalent cation represents one of psychiatry's most important treatments. Despite its demonstrated efficacy in reducing both the frequency and severity of recurrent affective episodes and decades of clinical use, the molecular mechanisms underlying its therapeutic actions have not fully been elucidated. In this report, we review the exciting recent progress in the identification of key components of signal transduction pathways (in particular, guanine nucleotide-binding proteins [G proteins], adenylyl cyclases, and protein kinase C isozymes) as targets for lithium's actions and attempt to integrate these effects with the large body of data emphasizing alterations in various neurotransmitter (particularly monoaminergic) systems. Regulation of signal transduction within critical regions of the brain by lithium affects the function of multiple neurotransmitter systems and may thus explain lithium's efficacy in protecting susceptible individuals from spontaneous, stress-induced, and drug-induced cyclic affective episodes. Recent evidence has also demonstrated significant effects of lithium on the regulation of gene expression in the central nervous system, effects that may play a major role in the long-term stabilization of mood. The identification of these intracellular targets for lithium's actions offers the potential for the development of novel, improved therapeutic agents and, in conjunction with molecular genetic approaches, may facilitate our understanding of the biological factors predisposing individuals to manic-depressive illness.

Objective: Several studies have suggested that many patients with anxiety disorders present in nonpsychiatric medical settings such as primary care facilities, emergency services, and general practice. This study examined the prevalence of panic disorder and generalized anxiety disorder among patients admitted to the general emergency service at an urban medical center in Israel., Methods: Four groups totaling 517 patients were assessed. The groups consisted of patients presenting with somatic complaints for whom no physical condition was diagnosed, patients with somatic complaints found to have a physical disorder, a group of nonpsychiatric consecutive admissions to the emergency service, and a group of referrals to the psychiatric emergency service., Results: The prevalence of panic disorder and generalized anxiety disorder in the entire sample was 2.7 percent, which is comparable to the prevalence rates reported in various community studies. However, the prevalence among patients with somatic complaints but no physical disorder was 6.7 percent, significantly higher than in the nonpsychiatric comparison groups. The prevalence in the group of psychiatric referrals was 4.8 percent., Conclusions: A population at risk for higher prevalence of anxiety disorders can be identified among patients seen in an emergency service. Physicians in primary care settings and general emergency services should consider anxiety disorders in the differential diagnosis of patients with somatic complaints but without a diagnosis of physical disorder.