An exploratory analysis of variance and volatility in epileptic electroencephalograms

The electroencephalogram (EEG) is a physiological time series that measures electrical activity at different locations in the brain, and plays an important role in epilepsy research. Exploring the variance and/or volatility may yield insights for seizure prediction, seizure detection and seizure propagation/dynamics.^ Maximal Overlap Discrete Wavelet Transforms (MODWTs) and ARMA-GARCH models were used to determine variance and volatility characteristics of 66 channels for different states of an epileptic EEG – sleep, awake, sleep-to-awake and seizure. The wavelet variances, changes in wavelet variances and volatility half-lives for the four states were compared for possible differences between seizure and non-seizure channels.^ The half-lives of two of the three seizure channels were found to be shorter than all of the non-seizure channels, based on 95% CIs for the pre-seizure and awake signals. No discernible patterns were found the wavelet variances of the change points for the different signals. ^

Genetic underpinnings of variation in brain ventricular volume: A genome-wide association study

The ventricular system is a critical component of the central nervous system (CNS) that is formed early in the developmental stages and remains functional through the lifetime. Changes in the ventricular system can be easily discerned via neuroimaging procedures and most of the time it reflects changes in the physiology of the CNS. In this study we attempted to identify specific genes associated with variation in ventricular volume in humans. Methods. We conducted a genome wide association (GWA) analysis of the volume of the lateral ventricles among 1605 individuals of European ancestry from two community based cohorts, the Genetics of Microangiopathic Brain Injury (GMBI; N=814) and Atherosclerosis Risk in Communities (ARIC; N=791). Significant findings from the analysis were tested for replication in both the cohorts and then meta-analyzed to get an estimate of overall significance. Results. In our GWA analyses, no single nucleotide polymorphism (SNP) reached a genome-wide significance of p<10−8. There were 25 SNPs in GMBI and 9 SNPs in ARIC that reached a threshold of p<10 −5. However, none of the top SNPs from each cohort were replicated in the other. In the meta-analysis, no SNP reached the genome-wide threshold of 5×10−8, but we identified five novel SNPs associated with variation in ventricular volume at the p<10 −5 level. Strongest association was for rs2112536 in an intergenic region on chromosome 5q33 (Pmeta= 8.46×10−7 ). The remaining four SNPs were located on chromosome 3q23 encompassing the gene for Calsyntenin-2 (CLSTN2). The SNPs with strongest association in this region were rs17338555 (Pmeta= 5.28×10 −6), rs9812091 (Pmeta= 5.89×10−6 ), rs9812283 (Pmeta= 5.97×10−6) and rs9833213 (Pmeta= 6.96×10−6). Conclusions. This GWA study of ventricular volumes in the community-based cohorts of European descent identifies potential locus on chromosomes 3 and 5. Further characterization of these loci may provide insights into pathophysiology of ventricular involvement in various neurological diseases.^

Characterization of the interaction between local cerebral metabolic rate for glucose and acid -base index in ischemic rat brain employing a double-isotope methodology

The association between increases in cerebral glucose metabolism and the development of acidosis is largely inferential, based on reports linking hyperglycemia with poor neurological outcome, lactate accumulation, and the severity of acidosis. We measured local cerebral metabolic rate for glucose (lCMRglc) and an index of brain pH--the acid-base index (ABI)--concurrently and characterized their interaction in a model of focal cerebral ischemia in rats in a double-label autoradiographic study, using ($\sp{14}$C) 2-deoxyglucose and ($\sp{14}$C) dimethyloxazolidinedione. Computer-assisted digitization and analysis permitted the simultaneous quantification of the two variables on a pixel-by-pixel basis in the same brain slices. Hemispheres ipsilateral to tamponade-induced middle cerebral occlusion showed areas of normal, depressed and elevated glucose metabolic rate (as defined by an interhemispheric asymmetry index) after two hours of ischemia. Regions of normal glucose metabolic rate showed normal ABI (pH $\pm$ SD = 6.97 $\pm$ 0.09), regions of depressed lCMRglc showed severe acidosis (6.69 $\pm$ 0.14), and regions of elevated lCMRglc showed moderate acidosis (6.88 $\pm$ 0.10), all significantly different at the.00125 level as shown by analysis of variance. Moderate acidosis in regions of increased lCMRglc suggests that anaerobic glycolysis causes excess protons to be generated by the uncoupling of ATP synthesis and hydrolysis. ^

The role of the intracellular second messenger cAMP in the induction of long-term morphological changes in sensory neurons of {\it Aplysia\/}

The present work examines the role of cAMP in the induction of the type of long-term morphological changes that have been shown to be correlated with long-term sensitization in Aplysia.^ To examine this issue, cAMP was injected into individual tail sensory neurons in the pleural ganglion to mimic, at the single cell level, the effects of behavioral training. After a 22 hr incubation period, the same cells were filled with horseradish peroxidase and 2 hours later the tissue was fixed and processed. Morphological analysis revealed that cAMP induced an increase in two morphological features of the neurons, varicosities and branch points. These structural alterations, which are similar to those seen in siphon sensory neurons of the abdominal ganglion following long-term sensitization training of the siphon-gill withdrawal reflex, could subserve the altered behavioral response of the animal. These results expose another role played by cAMP in the induction of learning, the initiation of a structural substrate, which, in concert with other correlates, underlies learning.^ cAMP was injected into sensory neurons in the presence of the reversible protein synthesis inhibitor, anisomycin. The presence of anisomycin during and immediately following the nucleotide injection completely blocked the structural remodeling. These results indicate that the induction of morphological changes by cAMP is a process dependent on protein synthesis.^ To further examine the temporal requirement for protein synthesis in the induction of these changes, the time of anisomycin exposure was varied. The results indicate that the cellular processes triggered by cAMP are sensitive to the inhibition of protein synthesis for at least 7 hours after the nucleotide injection. This is a longer period of sensitivity than that for the induction of another correlate of long-term sensitization, facilitation of the sensory to motor neuron synaptic connection. Thus, these findings demonstrate that the period of sensitivity to protein synthesis inhibition is not identical for all correlates of learning. In addition, since the induction of the morphological changes can be blocked by anisomycin pulses administered at different times during and following the cAMP injection, this suggests that cAMP is triggering a cascade of protein synthesis, with successive rounds of synthesis being dependent on successful completion of preceding rounds. Inhibition at any time during this cascade can block the entire process and so prevent the development of the structural changes.^ The extent to which cAMP can mimic the structural remodeling induced by long-term training was also examined. Animals were subjected to unilateral sensitization training and the morphology of the sensory neurons was examined twenty-four hours later. Both cAMP injection and long-term training produced a twofold increase in varicosities and approximately a fifty percent increase in the number of branch points in the sensory neuron arborization within the pleural ganglion. (Abstract shortened by UMI.) ^

Modulation of high voltage -activated calcium channels by phosphorylation

High voltage-activated (HVA) calcium channels from rat brain and rabbit heart are expressed in Xenopus laevis oocytes and their modulation by protein kinases studied. A subtype of the HVA calcium current expressed by rat brain RNA is potentiated by the phospholipid- and calcium-dependent protein kinase (PKC). The calcium channel clone $\alpha\sb{\rm1C}$ from rabbit heart is modulated by the cAMP-dependent protein kinase (PKA), and another factor present in the cytoplasm.^ The HVA calcium channels from rat brain do not belong to the L-type subclass since they are insensensitive to dihydropyridine (DHP) agonists and antagonists. The expressed currents do contain a N-type fraction which is identified by inactivation at depolarized potentials, and a P-type fraction as defined by blockade by the venom of the funnel web spider Agelenopsis Aperta. A non N-type fraction of this current is potentiated, by using phorbol esters to activate PKC. This residual fraction of current resembles the newly described Q-type channel from cerebellar granule cells in its biophysical properties, and potentiation by activation of PKC.^ The $\alpha\sb{\rm1C}$ clone from rabbit heart is expressed in oocytes and single-channel currents are measured using the cell-attached and cell-excised patch clamp technique. The single-channel current runs down within two minutes after patch excision into normal saline bath solution. The catalytic subunit of PKA + MgATP is capable of reversing this rundown for over 15 minutes. There also appears to be an additional factor present in the cytoplasm necessary for channel activity as revealed in experiments where PKA failed to prevent rundown.^ These data are important in that these types of channels are involved in synaptic transmission at many different types of synapses. The mammalian synapse is not accessible for these types of studies, however, the oocyte expression system allows access to HVA calcium channels for the study of their modulation by phosphorylation. ^

Functional studies of {\it twist\/} during mouse embryogenesis

A fundamental task in developmental biology is to understand the molecular mechanisms governing early embryogenesis. The aim of this study was to understand the developmental role of a putative basic helix-loop-helix (b-HLH) transcription factor, twist, during mouse embryogenesis.^ twist was originally identified in Drosophila as one of the zygotic genes, including snail, that were required for dorsal-ventral patterning. In Drosophila embryogenesis, twist is expressed in the cells of the ventral midline destined to form mesoderm. In embryos lacking twist expression, their ventral cells fail to form a ventral furrow and subsequently no mesoderm is formed.^ During mouse embryogenesis, twist is expressed after initial mesoderm formation in both mesoderm and cranial neural crest cell derivatives. To study the role of twist in vivo, twist-null embryos were generated by gene targeting. Embryos homozygous for the twist mutation die at midgestation. The most prominent phenotype in the present study was a failure of the cranial neural tube to close (exencephaly). twist-null embryos also showed defects in head mesenchyme, branchial arches, somites, and limb buds.^ To understand whether twist functions cell-autonomously and to investigate how twist-null cells interact with wild-type cells in vivo, twist chimeras composed of both twist-null and wild-type cells marked by the expression of the lacZgene were generated. Chimeric analysis revealed a correlation between the incidence of exencephaly and the contribution of the underlying twist-null head mesenchyme, thus strongly suggesting that twist-expressing head mesenchyme is required for the closure of the cranial neural tube. These studies have identified twist as a critical regulator for the mesenchymal fate determination within the cranial neural crest lineage. Most strikingly, twist-null head mesenchyme cells were always segregated from wild-type cells, indicating that the twist mutation altered the adhesive specificity of these cells. Furthermore, these results also indicated that twist functions cell-autonomously in the head, arch, and limb mesenchyme but non-cell-autonomously in the somites. Taken together, these studies have established the essential role of twist during mouse embryogenesis. ^

Calcium-dependent mechanisms and long-term potentiation: Role of NMDA receptors, voltage -dependent calcium channels and persistent protein kinase activity

Long-term potentiation (LTP) is a rapidly induced and long lasting increase in synaptic strength and is the leading cellular model for learning and memory in the mammalian brain. LTP was first identified in the hippocampus, a structure implicated in memory formation. LTP induction is dependent on postsynaptic Ca2+ increases mediated by N-methyl-D-aspartate (NMDA) receptors. Activation of other postsynaptic routes of Ca2+ entry, such as voltage-dependent Ca2+ channels (VDCCs) have subsequently been shown to induce a long-lasting increase in synaptic strength. However, it is unknown if VDCC-induced LTP utilized similar cellular mechanisms as the classical NMDA receptor-dependent LTP and if these two forms of LTP display similar properties. This dissertation determines the similarities and differences in VDCC and NMDA receptor-dependent LTP in area CA1 of hippocampal slices and demonstrates that VDCCs and NMDA receptors activate similar cellular mechanisms, such as protein kinases, to induce LTP. However, VDCC and NMDA receptor activated LTP induction mechanisms are compartmentalized in the postsynaptic neuron, such that they do not interact. Consistent with activation properties of NMDA receptors and VDCCs, NMDA receptor and VDCC-dependent LTP have different induction properties. In contrast to NMDA-dependent LTP, VDCC-induced potentiation does not require evoked presynaptic stimulation or display input specificity. These results indicate that there are two different routes of postsynaptic Ca2+ which can induce LTP and the compartmentation of VDCCs and NMDA receptors and/or their resulting Ca2+ increases may account for the distinction between these LTP induction mechanisms.^ One of the molecular targets for postsynaptic Ca2+ that is required for the induction of LTP is protein kinases. Evidence for the role of protein kinase activity in LTP expression is either correlational or controversial. We have utilized a broad range and potent inhibitors of protein kinases to systematically examine the temporal requirement for protein kinases in the induction and expression of LTP. Our results indicate that there is a critical period of persistent protein kinase activity required for LTP induction activated by tetanic stimulation and extending until 20 min after HFS. In addition, our results suggest that protein kinase activity during and immediately after HFS is not sufficient for LTP induction. These results provide evidence for persistent and/or Ca2+ independent protein kinase activity involvement in LTP induction. ^

Specificity of somatostatin receptor and G -protein interactions as characterized by somatostatin receptor subtype specific antibodies

The neuropeptide somatostatin is a widely distributed general inhibitor of endocrine, exocrine, gastrointestinal and neural functions. The biological actions of somatostatin are initiated by interaction with high affinity, plasma membrane somatostatin receptors (sst receptors). Five sst receptor subtypes have been cloned and sequence analysis shows they are all members of the G protein coupled receptor superfamily. The G proteins play a pivotal role in sst receptor signal transduction and the specificity of somatostatin receptor-G protein coupling defines the possible range of cellular responses. However, the data for endogenous sst receptor and G protein coupling is very limited, and even when it is available, the sst receptor subtypes involved in G protein coupling and signal transduction are unknown due to the expression of multiple sst receptor subtypes in target cell lines or tissues of somatostatin.^ In an effort to characterize each individual sst receptor subtypes, antisera against unique C-terminal regions of different sst receptor subtypes have been developed in our lab. In this report, antisera made against the sst1, sst2A and sst4 receptors are characterized. They are highly specific to their corresponding receptors and efficiently immunoprecipitate the sst receptors. Using these antibodies, the cell lines expressing these sst receptor subtypes were identified with both immunoprecipitation and Western blot methods. The development of sst receptor subtype specific antibodies make it possible to determine the specificity of the sst receptor subtype and G protein coupling in target cells or tissues expressing multiple sst receptors, two questions were addressed by this thesis: (1) whether different cellular environments affect receptor subtype and G protein coupling; (2) whether different sst receptors couple to different G proteins in similar cellular environments.^ Taken together our findings, both sst1 and sst2A receptors couple with G$\alpha\sb{\rm i1},$ G$\alpha\sb{\rm i2}$ and G$\alpha\sb{\rm i3}$ in CHO cells, G$\alpha\sb{\rm i2}$ and G$\alpha\sb{\rm i3}$ in GH$\sb4$C$\sb1$ cells. Further, sst2A receptors couple with G$\alpha\sb{\rm i1},$ G$\alpha\sb{\rm i2}$ and G$\alpha\sb{\rm i3}$ in AR4-2J cells while sst4 receptors couple with G$\alpha\sb{\rm i2}$ and G$\alpha\sb{\rm i3}$ in CHO cells. Therefore, the G protein coupling of the same sst receptors in different cell lines is basically similar in that they all couple with multiple $\alpha$-subunits of the G$\rm \sb{i}$ proteins, suggesting cellular environment has little effect on receptor and G protein coupling. Moreover, different sst receptors have similar G protein coupling specificities in the same cell line, suggesting components other than receptor and G$\alpha$ subunits in the signal transduction pathways may contribute to specific functions of each sst receptor subtype. This series of experiments represent a novel approach in dissecting signal transduction pathways and may have general application in the field. Furthermore, this is the first systematic study of sst receptor subtype and G protein $\alpha$-subunit interaction in both transfected cells and in normal cell lines. The information generated will be very useful in our understanding of sst receptor signal transduction pathways and in directing future sst receptor research. ^

Neocortical hyperexcitability defect in a mutant mouse model of spike-wave epilepsy, {\it stargazer}

Single-locus mutations in mice can express epileptic phenotypes and provide critical insights into the naturally occurring defects that alter excitability and mediate synchronization in the central nervous system (CNS). One such recessive mutation (on chromosome (Chr) 15), stargazer(stg/stg) expresses frequent bilateral 6-7 cycles per second (c/sec) spike-wave seizures associated with behavioral arrest, and provides a valuable opportunity to examine the inherited lesion associated with spike-wave synchronization.^ The existence of distinct and heterogeneous defects mediating spike-wave discharge (SWD) generation has been demonstrated by the presence of multiple genetic loci expressing generalized spike-wave activity and the differential effects of pharmacological agents on SWDs in different spike-wave epilepsy models. Attempts at understanding the different basic mechanisms underlying spike-wave synchronization have focused on $\gamma$-aminobutyric acid (GABA) receptor-, low threshold T-type Ca$\sp{2+}$ channel-, and N-methyl-D-aspartate receptor (NMDA-R)-mediated transmission. It is believed that defects in these modes of transmission can mediate the conversion of normal oscillations in a trisynaptic circuit, which includes the neocortex, reticular nucleus and thalamus, into spike-wave activity. However, the underlying lesions involved in spike-wave synchronization have not been clearly identified.^ The purpose of this research project was to locate and characterize a distinct neuronal hyperexcitability defect favoring spike-wave synchronization in the stargazer brain. One experimental approach for anatomically locating areas of synchronization and hyperexcitability involved an attempt to map patterns of hypersynchronous activity with antibodies to activity-induced proteins.^ A second approach to characterizing the neuronal defect involved examining the neuronal responses in the mutant following application of pharmacological agents with well known sites of action.^ In order to test the hypothesis that an NMDA receptor mediated hyperexcitability defect exists in stargazer neocortex, extracellular field recordings were used to examine the effects of CPP and MK-801 on coronal neocortical brain slices of stargazer and wild type perfused with 0 Mg$\sp{2+}$ artificial cerebral spinal fluid (aCSF).^ To study how NMDA receptor antagonists might promote increased excitability in stargazer neocortex, two basic hypotheses were tested: (1) NMDA receptor antagonists directly activate deep layer principal pyramidal cells in the neocortex of stargazer, presumably by opening NMDA receptor channels altered by the stg mutation; and (2) NMDA receptor antagonists disinhibit the neocortical network by blocking recurrent excitatory synaptic inputs onto inhibitory interneurons in the deep layers of stargazer neocortex.^ In order to test whether CPP might disinhibit the 0 Mg$\sp{2+}$ bursting network in the mutant by acting on inhibitory interneurons, the inhibitory inputs were pharmacologically removed by application of GABA receptor antagonists to the cortical network, and the effects of CPP under 0 Mg$\sp{2+}$aCSF perfusion in layer V of stg/stg were then compared with those found in +/+ neocortex using in vitro extracellular field recordings. (Abstract shortened by UMI.) ^

Inactivation and self -association of CaM kinase: Effects of ATP, substrate binding domains, and isozymic forms

Calcium/calmodulin-dependent protein kinase II (CaM kinase) is a multifunctional Ser/Thr protein kinase, that is highly enriched in brain and is involved in regulating many aspects of neuronal function. We observed that forebrain CaM kinase from crude homogenates, cytosolic fractions and purified preparations inactivates and translocates into the particulate fraction following autophosphorylation. Using purified forebrain CaM kinase as well as recombinant $\alpha$ isozyme, we determined that the formation of particulate enzyme was due to enzyme self-association. The conditions of autophosphorylation determine whether enzyme self-association and/or inactivation will occur. Self-association of CaM kinase is sensitive to pH, ATP concentration, and enzyme autophosphorylation. This process is prevented by saturating concentrations of ATP. However, in limiting ATP, pH is the dominant factor, and enzyme self-association occurs at pH values $\rm{<}7.0.$ Site-specific mutants were produced by substituting Ala for Thr286, Thr253, or Thr305,306 to determine whether these sites of autophosphorylation affect enzyme inactivation and self-association. The only mutation that influenced these processes was Ala286, which removed the protective effect afforded by autophosphorylation in saturating ATP. Enzyme inactivation occurs in the presence and absence of self-association and appears predominantly sensitive to nucleotide concentration, because saturating concentrations of $\rm Mg\sp{2+}/ADP$ or $\rm Mg\sp{2+}/ATP$ prevent this process. These data implicate the ATP binding pocket in both inactivation and self-association. We also observed that select peptide substrates and peptide inhibitors modeled after the autoregulatory domain of CaM kinase prevented these processes. The $\alpha$ and $\beta$ isozymes of CaM kinase were characterized independently, and were observed to exhibit differences in both enzyme inactivation and self-association. The $\beta$ isozyme was less sensitive to inactivation, and was never observed to self-associate. Biophysical characterization, and transmission electron microscopy coupled with image analysis indicated both isozymes were multimeric, however, the $\alpha$ and $\beta$ isozymes appeared structurally different. We hypothesize that the $\alpha$ subunit of CaM kinase plays both a structural and enzymatic role, and the $\beta$ subunit plays an enzymatic role. The ramifications for the functional differences observed for inactivation and self-association are discussed based on potential structural differences and autoregulation of the $\alpha$ and $\beta$ isozymes in both calcium-induced physiological and pathological processes. ^

Development of combined micro -preparation, separation, and mass spectrometry methods and application in the microdialysis study of neuropeptide metabolism

Two sets of mass spectrometry-based methods were developed specifically for the in vivo study of extracellular neuropeptide biochemistry. First, an integrated micro-concentration/desalting/matrix-addition device was constructed for matrix-assisted laser desorption ionization mass spectrometry (MALDI MS) to achieve attomole sensitivity for microdialysis samples. Second, capillary electrophoresis (CE) was incorporated into the above micro-liquid chromatography (LC) and MALDI MS system to provide two-dimensional separation and identification (i.e. electrophoretic mobility and molecular mass) for the analysis of complex mixtures. The latter technique includes two parts of instrumentation: (1) the coupling of a preconcentration LC column to the inlet of a CE capillary, and (2) the utilization of a matrix-precoated membrane target for continuous CE effluent deposition and for automatic MALDI MS analysis (imaging) of the CE track.^ Initial in vivo data reveals a carboxypeptidase A (CPA) activity in rat brain involved in extracellular neurotensin metabolism. Benzylsuccinic acid, a CPA inhibitor, inhibited neurotensin metabolite NT1-12 formation by 70%, while inhibitors of other major extracellular peptide metabolizing enzymes increased NT1-12 formation. CPA activity has not been observed in previous in vitro experiments. Next, the validity of the methodology was demonstrated in the detection and structural elucidation of an endogenous neuropeptide, (L)VV-hemorphin-7, in rat brain upon ATP stimulation. Finally, the combined micro-LC/CE/MALDI MS was used in the in vivo metabolic study of peptide E, a mu-selective opioid peptide with 25 amino acid residues. Profiles of 88 metabolites were obtained, their identity being determined by their mass-to-charge ratio and electrophoretic mobility. The results indicate that there are several primary cleavage sites in vivo for peptide E in the release of its enkephalin-containing fragments. ^

Structural determinants of subunit -subunit interactions and subcellular localization of the calcium /calmodulin -dependent protein kinase II

The multifunctional Ca$\sp{2+}$/calmodulin-dependent protein kinase II (CaM kinase) is a Ser/Thr directed protein kinase that participates in diverse Ca$\sp{2+}$ signaling pathways in neurons. The function of CaM kinase depends upon the ability of subunits to form oligomers and to interact with other proteins. Oligomerization is required for autophosphorylation which produces significant functional changes that include Ca$\sp{2+}$/calmodulin-independent activity and calmodulin trapping. Associations with other proteins localize CaM kinase to specific substrates and effectors which serves to optimize the efficiency and speed of signal transduction. In this thesis, we investigate the interactions that underlie the appropriate positioning of CaM kinase activity in cells. We demonstrate that the subcellular distribution of CaM kinase is dynamic in hippocampal slices exposed to anoxic/aglycemic insults and to high K$\sp{+}$-induced depolarization. We determine the localization of CaM kinase domains expressed in neurons and PC-12 cells and find that the C-terminal domain of the $\alpha$ subunit is necessary for localization to dendrites. Moreover, monomeric forms of the enzyme gain access to the nucleus. Attempts made to identify novel CaM kinase binding proteins using the yeast two-hybrid system resulted in the isolation of hundreds of positive clones. Those that have been sequenced are identical to CaM kinase isoforms. Finally, we report the discovery of specific regions within the C-terminal domain that are necessary and sufficient for subunit-subunit interactions. Differences between the $\alpha$ and $\beta$ isoforms were discovered that indicate unique structural requirements for oligomerization. A model for how CaM kinase subunits interact to form holoenzymes and how structural heterogeneity might influence CaM kinase function is presented. ^

The cGMP -PKG pathway is critical for inducing long-term sensitization of identified nociceptive sensory neurons

In various species, peripheral injury produces long-lasting sensitization of central and peripheral neurons representing the affected area. In Aplysia, memory-like traces (lasting days or weeks) of noxious peripheral stimulation include enhancement of central synaptic transmission and enhanced excitability of the central soma and peripheral branches of nociceptive sensory neurons. An important role for the cAMP-PKA-CREB pathway in consolidating long-term memory and inducing transcription-dependent synaptic potentiation has also been indicated by studies in rodents and Drosophila. ^ Much less attention has been paid to the cGMP-PKG pathway for transcription-dependent plasticity. Nevertheless, the cGMP-PKG pathway has been implicated in activity-dependent neural alterations lasting hours, and may trigger some forms of persistent pain. Recent evidence indicates PKG can regulate gene expression in the brain and several properties make it an attractive candidate for inducing long-term memories. ^ This dissertation reports that brief, noxious stimulation of a behaving, semi-intact preparation from mollusc, Aplysia californica, produces transcription-dependent, long-term hyperexcitability (LTH) of nociceptive sensory neurons that requires a nitric oxide (NO)-cGMP-protein kinase G (PKG) pathway and which lasts for at least 24 hours. Intracellular injection of cGMP is sufficient to induce LTH. Similarly, body wall injury induces LTH which can be blocked with specific inhibitors of the NO-cGMP-PKG pathway such as L-NMMA, ODQ, Rp-8-cGMPS, PKI-G and KT5823 by isolated perfusion of pleural ganglion sensory cells in or directly by intracellular injection. In contrast, specific inhibitors of the cAMP-PKA pathway (Rp-8-cAMPS, PKI-A and H-89) failed to block injury-induced LTH. Interestingly, co-injection of the cAMP-responsive element (CRE) blocked the induction of both cAMP and injury-induced LTH, but not cGMP-induced LTH. Furthermore, co-injection of cAMP and cGMP with the Ca2+ buffer BAPTA in reduced Ca2+ seawater blocked cAMP-, but not cGMP-induced LTH. These findings demonstrate that the NO-cGMP-PKG pathway and at least one other pathway (perhaps mediated by Ca2+), but not the cAMP-PKA pathway, are critical for inducing LTH during transient, noxious stimulation.^

Proteolytic mechanisms of cell injury following glucose -oxygen deprivation in primary neuronal cultures

Researchers have historically emphasized the contribution of caspase-3 to apoptotic but not necrotic cell death, while calpain has been implicated primarily in necrosis and, to a lesser extent, in apoptosis. Activation of these proteases occurs in vivo following various CNS insults including ischemia. In addition, both necrotic and apoptotic cell death phenotypes are detected following ischemia. However, the contributions of calpain and caspase-3 to apoptotic and necrotic cell death phenotypes following CNS insults are relatively unexplored. To date, no study has examined the concurrent activation of calpain and caspase-3 in necrotic and apoptotic cell death phenotypes following any CNS insult. The present study employed oxygen-glucose deprivation (OGD) to determine the relative contributions of caspase-3 and calpain to apoptotic and necrotic cell death following OGD. Experiments characterized a model of OGD by evaluating cell viability and characterizing the cell death phenotypes following OGD in primary septo-hippocampal co-cultures. Furthermore, cell markers (NeuN and MAP2 or GFAP) assessed the effects of OGD on neuronal and astroglial viability, respectively. In addition, calpain and caspase-3 mediated proteolysis of α-spectrin was examined using Western blot techniques. Activation of these proteases in individual cells phenotypically characterized as apoptotic and necrotic was also evaluated by using antibodies specific for calpain or caspase-3 mediated breakdown products to α-spectrin. Administration of appropriate caspase-3 and calpain inhibitors also examined the effects of protease inhibition on cell death. OGD produced prominent expression of apoptotic cell death phenotypes primarily in neurons, with relatively little damage to astroglia. Although Western blot data suggested greater proteolysis of α-spectrin by calpain than caspase-3, co-activation of both proteases was usually detected in cells exhibiting apoptotic or necrotic cell death phenotypes. While inhibition of calpain and caspase-3 activity decreased LDH release following OGD, it was not clear whether this effect was also associated with a decrease in cell death and the appearance of apoptotic cell death phenotypes. These data demonstrate that both calpain and caspase-3 contribute to the expression of apoptotic cell death phenotypes following OGD, and that calpain could potentially have a larger role in the expression of apoptotic cell death than previously thought. ^

Predictors of benzodiazepine self -administration behavior in anxious patients

The purpose of this study was to examine factors that may be associated with benzodiazepine (BZ) self-administration and risks of dependence in anxious patients. Preliminary work included examination of psychosocial characteristics and subjective drug response as potential predictors of medication use. Fifty-five M, F patients with generalized anxiety or panic disorder participated in a 3-week outpatient Choice Procedure in which they self-medicated “as needed” with alprazolam (Alz) and placebo. Findings showed that a large amount of variance in alprazolam preference, frequency, and quantity of use could be predicted by measures of anxiety, drug liking, and certain personality characteristics. The primary study extended this work by examining whether individual differences in Alz sensitivity also predict patterns of use. Twenty anxious patients participated in the study, which required 11 weekly clinic visits. Ten of these also participated in a baseline assessment of HPA-axis function that involved 24-hour monitoring of cortisol and ACTH levels and a CRH Stimulation Test. This assessment was conducted on the basis of prior evidence that steroid metabolites exert neuromodulatory effects on the GABA A receptor and that HPA-axis function may be related to BZ sensitivity and long-term disability in anxious patients. Patients were classified as either HIGH or LOW users based on their p.r.n. patterns of Alz use during the first 3 weeks of the study. They then participated in a 4-week dose response trial in which they received prescribed doses of medication (placebo, 0.25, 0.5, and 1.0mg Alz), each taken TID for 1 week. The dose response trial was followed by a second 3-week Choice Procedure. Findings were not indicative of biological differences in Alz sensitivity between the HIGH and LOW users. However, the HIGH users had higher baseline anxiety and greater anxiolytic response to Alz than the LOW users. Anxiolytic benefits of p.r.n. and prescribed dosing were shown to be comparable, and patients' conservative patterns of p.r.n. medication use were not affected by the period of prescribed dosing. Although there was not strong evidence to suggest relationships between HPA-axis function and Alz use or sensitivity, interesting findings emerged about the relationship between HPA-axis function and anxiety. ^