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Pathogenic mutations in LRRK2 cause Parkinson's disease (PD). The G2019S variant is the most common, which results in abnormally high kinase activity. Compounds that target LRRK2 kinase activity are currently being developed and tested in clinical trials. We recently found that G2019S LRRK2 causes mitochondrial DNA (mtDNA) damage and treatment with multiple classes of LRRK2 kinase inhibitors at concentrations associated with dephosphorylation of LRRK2 reversed mtDNA damage to healthy control levels. Because maintaining the normal function of LRRK2 in heterozygous G2019S LRRK2 carriers while specifically targeting the G2019S LRRK2 activity could have an advantageous safety profile, we explored the efficacy of a G2019S mutant selective LRRK2 inhibitor to reverse mtDNA damage in G2019S LRRK2 models and patient cells relative to non-selective LRRK2 inhibitors. Potency of LRRK2 kinase inhibition by EB-42168, a G2019S mutant LRRK2 kinase inhibitor, and MLi-2, a non-selective inhibitor, was determined by measuring phosphorylation of LRRK2 at Ser935 and/or Ser1292 using quantitative western immunoblot analysis. The Mito DNA DX assay, which allows for the accurate real-time quantification of mtDNA damage in a 96-well platform, was performed in parallel. We confirmed that EB-42168 selectively inhibits LRRK2 phosphorylation on G2019S LRRK2 relative to wild-type LRRK2. On the other hand, MLi-2 was equipotent for wild-type and G2019S LRRK2. Acute treatment with EB-42168 inhibited LRRK2 phosphorylation and also restored mtDNA damage to healthy control levels. We further investigated the relationship between LRRK2 kinase activity, mtDNA damage and mitophagy. Levels of mtDNA damage caused by G2019S LRRK2 were fully re-established within 2 h of a LRRK2 inhibitor wash out and recovery experiment, indicating the mtDNA damage phenotype is highly dynamic. G2019S LRRK2 mitophagy defects were not alleviated with LRRK2 kinase inhibition, suggesting that mitophagy is not mechanistically regulating LRRK2 kinase-mediated reversal of mtDNA damage in this acute timeframe. Abrogation of mtDNA damage with the mutant selective tool inhibitor EB-42168 demonstrates the potential of a precision medicine approach for LRRK2 G2019S PD. Levels of mtDNA damage may serve as a potential pharmacodynamic biomarker of altered kinase activity that could be useful for small molecule development and clinical trials., (© 2024. The Author(s).)

The G2019S variant of LRRK2, which causes an increase in kinase activity, is associated with the occurrence of Parkinson's disease (PD). Potent, mutation-selective, and brain penetrant inhibitors of LRRK2 can suppress the biological effects specific to G2019S-LRRK2 that cause pathogenicity. We report the discovery of a series of cyanoindane and cyanotetralin kinase inhibitors culminating in compound 34 that demonstrated selective inhibition of phosphorylation of LRRK2 in the mouse brain. These novel inhibitors may further enable the precision medicine path for future PD therapeutics., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Background: A common genetic mutation that causes Parkinson's disease (PD) is the G2019S LRRK2 mutation. A precision medicine approach that selectively blocks only excess kinase activity of the mutant allele could yield a safe and effective treatment for G2019S LRRK2 PD., Objective: To determine the activity of a G2019S mutant selective leucine-rich repeat kinase 2 (LRRK2) kinase inhibitor as compared to a nonselective inhibitor in blood of subjects with genetic and idiopathic PD on two LRRK2 biomarkers, pSer935 LRRK2 and pThr73 Rab10., Methods: Blood was collected from 13 subjects with or without a G2019S LRRK2 mutation with PD and one healthy control. Peripheral blood mononuclear cells were treated ex vivo with a novel G2019S LRRK2 inhibitor (EB-42168) or the nonselective inhibitor MLi-2. Quantitative western immunoblot analyses were performed., Results: EB-42168 was 100 times more selective for G2019S LRRK2 when compared to wild-type (WT) LRRK2. Concentrations that inhibited phosphorylation of pSer935 LRRK2 by 90% in homozygous G2019S LRRK2 patients, inhibited pSer935 LRRK2 by 36% in heterozygous patients, and by only 5% in patients carrying only the WT allele. Similar selectivity was seen for pThr73 Rab10. MLi-2 showed an equivalent level of inhibition across all genotypes., Conclusions: These findings demonstrate that EB-42168, a G2019S LRRK2 selective inhibitor, lowers mutant G2019S LRRK2 phosphorylated biomarkers while simultaneously sparing WT LRRK2. Selective targeting of G2019S LRRK2 with a small molecule lays the foundation for a precision medicine treatment of G2019S LRRK2 PD. © 2021 ESCAPE Bio, Inc. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society., (© 2021 ESCAPE Bio, Inc. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.)

Pathogenic variants in the leucine-rich repeat kinase 2 (LRRK2) gene have been identified that increase the risk for developing Parkinson's disease in a dominantly inherited fashion. These pathogenic variants, of which G2019S is the most common, cause abnormally high kinase activity, and compounds that inhibit this activity are being pursued as potentially disease-modifying therapeutics. Because LRRK2 regulates important cellular processes, developing inhibitors that can selectively target the pathogenic variant while sparing normal LRRK2 activity could offer potential advantages in heterozygous carriers. We conducted a high-throughput screen and identified a single selective compound that preferentially inhibited G2019S-LRRK2. Optimization of this scaffold led to a series of novel, potent, and highly selective G2019S-LRRK2 inhibitors.

A common pathological hallmark of age-related neurodegenerative diseases is the intracellular accumulation of protein aggregates such as α-synuclein in Parkinson's disease, TDP-43 in ALS, and tau in Alzheimer's disease. Enhancing intracellular clearance of aggregation-prone proteins is a plausible strategy for slowing progression of neurodegenerative diseases and there is great interest in identifying molecular targets that control protein turnover. One of the main routes for protein degradation is through the proteasome, a multisubunit protease that degrades proteins that have been tagged with a polyubiquitin chain by ubiquitin activating and conjugating enzymes. Published data from cellular models indicate that Ubiquitin-specific protease 14 (USP14), a deubiquitinating enzyme (DUB), slows the degradation of tau and TDP-43 by the proteasome and that an inhibitor of USP14 increases the degradation of these substrates. We conducted similar experiments designed to evaluate tau, TDP-43, or α-synuclein levels in cells after overexpressing USP14 or knocking down endogenous expression by siRNA.

Autophagy is the primary process for recycling cellular constituents through lysosomal degradation. In addition to nonselective autophagic engulfment of cytoplasm, autophagosomes can recognize specific cargo by interacting with ubiquitin-binding autophagy receptors such as SQSTM1/p62 (sequestosome 1). This selective form of autophagy is important for degrading aggregation-prone proteins prominent in many neurodegenerative diseases. We carried out a high content image-based siRNA screen (4 to 8 siRNA per gene) for modulators of autophagic flux by monitoring fluorescence of GFP-SQSTM1 as well as colocalization of GFP-SQSTM1 with LAMP2 (lysosomal-associated membrane protein 2)-positive lysosomal vesicles. GFP-SQSTM1 and LAMP2 phenotypes of primary screen hits were confirmed in 2 cell types and profiled with image-based viability and MTOR signaling assays. Common seed analysis guided siRNA selection for these assays to reduce bias toward off-target effects. Confirmed hits were further validated in a live-cell assay to monitor fusion of autophagosomes with lysosomes. Knockdown of 10 targets resulted in phenotypic profiles across multiple assays that were consistent with upregulation of autophagic flux. These hits include modulators of transcription, lysine acetylation, and ubiquitination. Two targets, KAT8 (K[lysine] acetyltransferase 8) and CSNK1A1 (casein kinase 1, α 1), have been implicated in autophagic regulatory feedback loops. We confirmed that CSNK1A1 knockout (KO) cell lines have accelerated turnover of long-lived proteins labeled with (14)C-leucine in a pulse-chase assay as additional validation of our screening assays. Data from this comprehensive autophagy screen point toward novel regulatory pathways that might yield new therapeutic targets for neurodegeneration.

synGAP is a neuron-specific Ras and Rap GTPase-activating protein (GAP) found in high concentrations in the postsynaptic density (PSD) fraction from the mammalian forebrain. We have previously shown that, in situ in the PSD fraction or in recombinant form in Sf9 cell membranes, synGAP is phosphorylated by Ca(2+)/calmodulin-dependent protein kinase II (CaMKII), another prominent component of the PSD. Here, we show that recombinant synGAP (r-synGAP), lacking 102 residues at the N terminus, can be purified in soluble form and is phosphorylated by cyclin-dependent kinase 5 (CDK5) as well as by CaMKII. Phosphorylation of r-synGAP by CaMKII increases its HRas GAP activity by 25% and its Rap1 GAP activity by 76%. Conversely, phosphorylation by CDK5 increases r-synGAP's HRas GAP activity by 98% and its Rap1 GAP activity by 20%. Thus, phosphorylation by both kinases increases synGAP activity; CaMKII shifts the relative GAP activity toward inactivation of Rap1, and CDK5 shifts the relative activity toward inactivation of HRas. GAP activity toward Rap2 is not altered by phosphorylation by either kinase. CDK5 phosphorylates synGAP primarily at two sites, Ser-773 and Ser-802. Phosphorylation at Ser-773 inhibits r-synGAP activity, and phosphorylation at Ser-802 increases it. However, the net effect of concurrent phosphorylation of both sites, Ser-773 and Ser-802, is an increase in GAP activity. synGAP is phosphorylated at Ser-773 and Ser-802 in the PSD fraction, and its phosphorylation by CDK5 and CaMKII is differentially regulated by activation of NMDA-type glutamate receptors in cultured neurons., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Laboratory detection of nicotine exposure is important for establishing eligibility for organ transplant and elective surgery. Nicotine testing is also used to verify compliance with nicotine replacement therapies (NRT), smoking cessation programs and for life insurance purposes. Nicotine metabolites, such as cotinine and trans-3'-hydroxycotinine, are used as biomarkers of nicotine exposure. For some clinical applications, it is important to distinguish between active use of tobacco products versus NRT. Anabasine is a tobacco alkaloid that has been used as a biomarker of active tobacco use. However, the use of anabasine as an insecticide, and its presence in consumables other than nicotine products, suggests that anabasine may not be specific to tobacco use/exposure. Here, we determine the reference interval for anabasine in the urine of nonsmokers and compare it to the range of anabasine concentrations observed in the presence or absence of nicotine metabolites., (© The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Densin is an abundant scaffold protein in the postsynaptic density (PSD) that forms a high-affinity complex with αCaMKII and α-actinin. To assess the function of densin, we created a mouse line with a null mutation in the gene encoding it (LRRC7). Homozygous knock-out mice display a wide variety of abnormal behaviors that are often considered endophenotypes of schizophrenia and autism spectrum disorders. At the cellular level, loss of densin results in reduced levels of α-actinin in the brain and selective reduction in the localization of mGluR5 and DISC1 in the PSD fraction, whereas the amounts of ionotropic glutamate receptors and other prominent PSD proteins are unchanged. In addition, deletion of densin results in impairment of mGluR- and NMDA receptor-dependent forms of long-term depression, alters the early dynamics of regulation of CaMKII by NMDA-type glutamate receptors, and produces a change in spine morphology. These results indicate that densin influences the function of mGluRs and CaMKII at synapses and contributes to localization of mGluR5 and DISC1 in the PSD fraction. They are consistent with the hypothesis that mutations that disrupt the organization and/or dynamics of postsynaptic signaling complexes in excitatory synapses can cause behavioral endophenotypes of mental illness.

Brain injury, genetic manipulations, and pharmacological treatments can result in alterations of motor skills in mice. Fine motor coordination and balance can be assessed by the beam walking assay. The goal of this test is for the mouse to stay upright and walk across an elevated narrow beam to a safe platform. This test takes place over 3 consecutive days: 2 days of training and 1 day of testing. Performance on the beam is quantified by measuring the time it takes for the mouse to traverse the beam and the number of paw slips that occur in the process. Here we report the protocol used in our laboratory, and representative results from a cohort of C57BL/6 mice. This task is particularly useful for detecting subtle deficits in motor skills and balance that may not be detected by other motor tests, such as the Rotarod.

Sensitization to inflammatory pain is a pathological form of neuronal plasticity that is poorly understood and treated. Here we examine the role of the SH3 domain of postsynaptic density 95 (PSD95) by using mice that carry a single amino-acid substitution in the polyproline-binding site. Testing multiple forms of plasticity we found sensitization to inflammation was specifically attenuated. The inflammatory response required recruitment of phosphatidylinositol-3-kinase-C2alpha to the SH3-binding site of PSD95. In wild-type mice, wortmannin or peptide competition attenuated the sensitization. These results show that different types of behavioural plasticity are mediated by specific domains of PSD95 and suggest novel therapeutic avenues for reducing inflammatory pain.

The membrane-associated guanylate kinases (MAGUKs) PSD-95, PSD-93 and SAP102 are thought to have crucial roles in both AMPA receptor trafficking and formation of NMDA receptor-associated signalling complexes involved in synaptic plasticity. While PSD-95, PSD-93, and SAP102 appear to have similar roles in AMPA receptor trafficking, it is not known whether these MAGUKs also have functionally similar roles in synaptic plasticity. To explore this issue we examined several properties of basal synaptic transmission in the hippocampal CA1 region of PSD-93 and PSD-95 mutant mice and compared the ability of a number of different synaptic stimulation protocols to induce long-term potentiation (LTP) and long-term depression (LTD) in these mutants. We find that while both AMPA and NMDA receptor-mediated synaptic transmission are normal in PSD-93 mutants, PSD-95 mutant mice exhibit clear deficits in AMPA receptor-mediated transmission. Moreover, in contrast to the facilitation of LTP induction and disruption of LTD observed in PSD-95 mutant mice, PSD-93 mutant mice exhibit deficits in LTP and normal LTD. Our results suggest that PSD-95 has a unique role in AMPA receptor trafficking at excitatory synapses in the hippocampus of adult mice and indicate that PSD-93 and PSD-95 have essentially opposite roles in LTP, perhaps because these MAGUKs form distinct NMDA receptor signalling complexes that differentially regulate the induction of LTP by different patterns of synaptic activity.

SynGAP, a prominent Ras/Rap GTPase-activating protein in the postsynaptic density, regulates the timing of spine formation and trafficking of glutamate receptors in cultured neurons. However, the molecular mechanisms by which it does this are unknown. Here, we show that synGAP is a key regulator of spine morphology in adult mice. Heterozygous deletion of synGAP was sufficient to cause an excess of mushroom spines in adult brains, indicating that synGAP is involved in steady-state regulation of actin in mature spines. Both Ras- and Rac-GTP levels were elevated in forebrains from adult synGAP(+/-) mice. Rac is a well known regulator of actin polymerization and spine morphology. The steady-state level of phosphorylation of cofilin was also elevated in synGAP(+/-) mice. Cofilin, an F-actin severing protein that is inactivated by phosphorylation, is a downstream target of a pathway regulated by Rac. We show that transient regulation of cofilin by treatment with NMDA is also disrupted in synGAP mutant neurons. Treatment of wild-type neurons with 25 mum NMDA triggered transient dephosphorylation and activation of cofilin within 15 s. In contrast, neurons cultured from mice with a homozygous or heterozygous deletion of synGAP lacked the transient regulation by the NMDA receptor. Depression of EPSPs induced by a similar treatment of hippocampal slices with NMDA was disrupted in slices from synGAP(+/-) mice. Our data show that synGAP mediates a rate-limiting step in steady-state regulation of spine morphology and in transient NMDA-receptor-dependent regulation of the spine cytoskeleton.

At excitatory synapses onto hippocampal CA1 pyramidal cells, activation of cyclic AMP-dependent protein kinase and subsequent down-regulation of protein phosphatases has a crucial role in the induction of long-term potentiation by low-frequency patterns of synaptic stimulation. Because the second messenger cyclic guanosine 3',5'monophosphate can regulate the activity of different forms of the cyclic AMP degrading enzyme phosphodiesterase, we examined whether increases in cyclic guanosine 3',5'monophosphate can modulate long-term potentiation induction in the mouse hippocampal CA1 region through effects on cyclic AMP signaling. Using the cyclic guanosine 3',5'monophosphate-specific phosphodiesterase inhibitor zaprinast or the nitric oxide donor S-nitroso-D,L-penicillamine to elevate cyclic guanosine 3',5'monophosphate levels we found that increases in cyclic guanosine 3',5'monophosphate strongly inhibit the induction of long-term potentiation by low-frequency patterns of synaptic stimulation where protein kinase A activation is required for long-term potentiation induction. In contrast, zaprinast and S-nitroso-D,L-penicillamine had no effect on the induction of long-term potentiation by high-frequency patterns of synaptic stimulation that induce long-term potentiation in a protein kinase A-independent manner. Directly activating protein kinase A with the phosphodiesterase-resistant cyclic AMP analog 8-Br-cAMP, blocking all phosphodiesterases with 3-isobutyl-1-methylxanthine, or inhibiting protein phosphatases rescued long-term potentiation induction in zaprinast-treated slices. Together, these results suggest that increases in cyclic guanosine 3',5'monophosphate inhibit long-term potentiation by activating phosphodiesterases that interfere with the protein kinase A-mediated suppression of protein phosphatases needed for long-term potentiation induction. Consistent with the notion that this cyclic guanosine 3',5'monophosphate-mediated inhibitory pathway is recruited by some patterns of synaptic activity, blocking cyclic guanosine 3',5'monophosphate production strongly facilitated the induction of long-term potentiation by long trains of theta-frequency synaptic stimulation. Together, our results indicate that increases in cyclic guanosine 3',5'monophosphate can act as a long-term potentiation suppressor mechanism that selectively constrains the induction of protein kinase A-dependent forms of long-term potentiation induced by low-frequency patterns of synaptic stimulation.

Short-term and long-term changes in the strength of synapses in neural networks underlie working memory and long-term memory storage in the brain. These changes are regulated by many biochemical signalling pathways in the postsynaptic spines of excitatory synapses. Recent findings about the roles and regulation of the small GTPases Ras, Rap and Rac in spines provide new insights into the coordination and cooperation of different pathways to effect synaptic plasticity. Here, we present an initial working representation of the interactions of five signalling cascades that are usually studied individually. We discuss their integrated function in the regulation of postsynaptic plasticity.

Many forms of mental retardation and cognitive disability are associated with abnormalities in dendritic spine morphology. Visualization of spines using live-imaging techniques provides convincing evidence that spine morphology is altered in response to certain forms of LTP-inducing stimulation. Thus, information storage at the cellular level appears to involve changes in spine morphology that support changes in synaptic strength produced by certain patterns of synaptic activity. Because the structure of a spine is determined by its underlying actin cytoskeleton, there has been much effort to identify signaling pathways linking synaptic activity to control of actin polymerization. This review, part of the TINS Synaptic Connectivity series, discusses recent studies that implicate EphB and NMDA receptors in the regulation of actin-binding proteins through modulation of Rho family small GTPases.

An oft-overlooked consequence of fever is the occurrence of thermoregulatory heat-seeking/producing behaviors. Subdiaphragmatic vagotomy attenuates fever resulting from low dose, peripherally administered pyrogens, suggesting that the vagus is involved in generating the pathogen-induced rise in core body temperature (T(c)). This study was designed to confirm that rats utilize behavioral thermoregulation to augment fever following systemic administration of lipopolysaccharide (LPS), and to test the hypothesis that, in febrile animals, vagotomy would block the preference for a higher ambient temperature (T(a)) as T(c) is rising. First, female Sprague-Dawley rats received IP injections of either saline or LPS (50 microg/kg), prior to placement inside a thermal gradient that offered subjects T(a) values between 7 and 45 degrees C. LPS injection caused significant increases in T(c) and selection of a higher T(a) as compared to saline administration. Second, groups of rats were vagotomized, sham-vagotomized or received no surgery, and then underwent the same gradient testing procedure. Vagotomy attenuated LPS-induced fever, but did not influence the concomitant behavioral thermoregulatory response. All groups selected comparable, higher T(a) values following LPS vs. saline. These data suggest that the reduction in the febrile response to LPS administration following vagotomy is not due to inhibition of the behavioral thermoregulatory response to the pyrogen. Rather, this behavioral response to LPS appears to be mediated by a nonvagal mechanism.

At excitatory synapses, the postsynaptic scaffolding protein postsynaptic density 95 (PSD-95) couples NMDA receptors (NMDARs) to the Ras GTPase-activating protein SynGAP. The close association of SynGAP and NMDARs suggests that SynGAP may have an important role in NMDAR-dependent activation of Ras signaling pathways, such as the MAP kinase pathway, and in synaptic plasticity. To explore this issue, we examined long-term potentiation (LTP), p42 MAPK (ERK2) signaling, and spatial learning in mice with a heterozygous null mutation of the SynGAP gene (SynGAP(-/+)). In SynGAP(-/+) mutant mice, the induction of LTP in the hippocampal CA1 region was strongly reduced in the absence of any detectable alteration in basal synaptic transmission and NMDAR-mediated synaptic currents. Although basal levels of activated ERK2 were elevated in hippocampal extracts from SynGAP(-/+) mice, NMDAR stimulation still induced a robust increase in ERK activation in slices from SynGAP(-/+) mice. Thus, although SynGAP may regulate the ERK pathway, its role in LTP most likely involves additional downstream targets. Consistent with this, the amount of potentiation induced by stimulation protocols that induce an ERK-independent form of LTP were also significantly reduced in slices from SynGAP(-/+) mice. An elevation of basal phospho-ERK2 levels and LTP deficits were also observed in SynGAP(-/+)/H-Ras(-)/- double mutants, suggesting that SynGAP may normally regulate Ras isoforms other than H-Ras. A comparison of SynGAP and PSD-95 mutants suggests that PSD-95 couples NMDARs to multiple downstream signaling pathways with very different roles in LTP and learning.

Activation of metabotropic glutamate receptors (mGluRs) with the group I mGluR selective agonist (R,S)-3,5-dihydroxyphenylglycine (DHPG) induces a long-term depression (LTD) of excitatory synaptic transmission in the CA1 region of the hippocampus. Here we investigated the potential roles of pre- and postsynaptic processes in the DHPG-induced LTD at excitatory synapses onto hippocampal pyramidal cells in the mouse hippocampus. Activation of mGluRs with DHPG, but not ACPD, induced LTD at both Schaffer collateral/commissural fiber synapses onto CA1 pyramidal cells and at associational/commissural fiber synapses onto CA3 pyramidal cells. DHPG-induced LTD was blocked when the G-protein inhibitor guanosine-5'-O-(2-thiodiphosphate) was selectively delivered into postsynaptic CA1 pyramidal cells via an intracellular recording electrode, suggesting that DHPG depresses synaptic transmission through a postsynaptic, GTP-dependent signaling pathway. The effects of DHPG were also strongly modulated, however, by experimental manipulations that altered presynaptic calcium influx. In these experiments, we found that elevating extracellular Ca(2+) concentrations ([Ca(2+)](o)) to 6 mM almost completely blocked the effects of DHPG, whereas lowering [Ca(2+)](o) to 1 mM significantly enhanced the ability of DHPG to depress synaptic transmission. Enhancing Ca(2+) influx by prolonging action potential duration with bath applications of the K(+) channel blocker 4-aminopyridine (4-AP) also strongly reduced the effects of DHPG in the presence of normal [Ca(2+)](o) (2 mM). Although these findings indicate that alterations in Ca(2+)-dependent signaling processes strongly regulate the effects of DHPG on synaptic transmission, they do not distinguish between potential pre- versus postsynaptic sites of action. We found, however, that while inhibiting both pre- and postsynaptic K(+) channels with bath-applied 4-AP blocked the effects of DHPG; inhibition of postsynaptic K(+) channels alone with intracellular Cs(+) and TEA had no effect on the ability of DHPG to inhibit synaptic transmission. This suggests that presynaptic changes in transmitter release contribute to the depression of synaptic transmission by DHPG. Consistent with this, DHPG induced a persistent depression of both AMPA and N-methyl-D-aspartate receptor-mediated components of excitatory postsynaptic currents in voltage-clamped pyramidal cells. Together our results suggest that activation of postsynaptic mGluRs suppresses transmission at excitatory synapses onto CA1 pyramidal cells through presynaptic effects on transmitter release.

This experiment examined the effects of intraperitoneal (i.p.) clozapine (CLZ) compared to haloperidol (HAL) on operant responding for heat reinforcement in a cold environment (-8 degrees C). Three doses of CLZ (1, 3 and 5 mg/kg) were found to dose-dependently increase responding for heat while lowering core temperature (T(c)) only at the highest dose. Three doses of HAL (0.1, 0.3 and 0.5 mg/kg) dose-dependently decreased operant responding which resulted in a dose-dependent decrease in T(c). The highest dose of CLZ was then tested in two other paradigms: a reinforcement schedule in which heat was available as long as the lever was held down, and a temperature gradient (range 7-45 degrees C) in which access to heat required minimal motor effort. The ad libitum reinforcement schedule still did not provide enough heat to overcome the hypothermic effects of CLZ. However, in the gradient, rats receiving CLZ selected a warmer region of the gradient, and T(c) was higher than that of controls. These data support CLZ's reputation for having minimal motor side effects. Unlike HAL, the hypothermic effects of CLZ appear to be unrelated to effects of the drug on movement.

Cell lines are commonly used for studying recombinant heterooligomeric ion channels with defined subunit composition. Such studies often ignore the contribution of endogenous proteins in the assembly of mature channels. We examined whether an endogenous subunit was required for the functional expression of gamma-aminobutyric acid type A (GABA(A)) receptors in WSS-1 cells, HEK293 cells stably expressing recombinant alpha1 and gamma2 subunits. Our pharmacological and RT-PCR analyses of GABA(A) receptors and their mRNAs in WSS-1 cells confirm the presence of alpha1 and gamma2 subunits and suggest the existence of an endogenous beta3 subunit. Whole-cell GABA-evoked currents recorded from untransfected WSS-1 cells were blocked by bicuculline methiodide and enhanced by anesthetics and anticonvulsants including the subunit-selective compounds diazepam and loreclezole. These data suggest that, in addition to the gamma2 subunit, WSS-1 cell receptors also contain beta2/3 subunits. RT-PCR revealed that WSS-1 cells and parental HEK293 cells contain beta3 mRNA. We examined the contribution of the beta3 subunit in the function of receptors formed by expression of alpha1 and gamma2S subunits. Untransfected HEK293 cells were unresponsive to GABA. Cells transfected with alpha1 and gamma2S cDNAs displayed small diazepam and loreclezole responsive GABA-activated currents. By contrast, the expression of alpha1 and gamma2S cDNAs in the neuroblastoma NB41A3 cell line, that lacks beta subunit mRNAs, failed to produce functional receptors. These data reaffirm that alpha1 and gamma2S subunits alone do not form functional GABA(A) receptors and that receptors of WSS-1 cells contain alpha1, beta3 and gamma2S subunits.

The induction of long-term potentiation (LTP) and long-term depression (LTD) at excitatory synapses in the hippocampus can be strongly modulated by patterns of synaptic stimulation that otherwise have no direct effect on synaptic strength. Likewise, patterns of synaptic stimulation that induce LTP or LTD not only modify synaptic strength but can also induce lasting changes that regulate how synapses will respond to subsequent trains of stimulation. Collectively known as metaplasticity, these activity-dependent processes that regulate LTP and LTD induction allow the recent history of synaptic activity to influence the induction of activity-dependent changes in synaptic strength and may thus have an important role in information storage during memory formation. To explore the cellular and molecular mechanisms underlying metaplasticity, we investigated the role of metaplasticity in the induction of LTP by theta-frequency (5-Hz) synaptic stimulation in the hippocampal CA1 region. Our results show that brief trains of theta-frequency stimulation not only induce LTP but also activate a process that inhibits the induction of additional LTP at potentiated synapses. Unlike other forms of metaplasticity, the inhibition of LTP induction at potentiated synapses does not appear to arise from activity-dependent changes in NMDA receptor function, does not require nitric oxide signaling, and is strongly modulated by beta-adrenergic receptor activation. Together with previous findings, our results indicate that mechanistically distinct forms of metaplasticity regulate LTP induction and suggest that one way modulatory transmitters may act to regulate synaptic plasticity is by modulating metaplasticity.

A thermal gradient (temperature range 7-45 degrees C) was used to assess ambient temperature (Ta) preferences of rats following treatment with clonidine (25 microg/kg), norepinephrine (NE, 250 microg/kg), isoproterenol (ISO, 50 microg/kg), and ephedrine (EPH, 10 mg/kg). Clonidine produced a preference for a temperature (31.5 degrees C) slightly warmer than that preferred after saline (28.3 degrees C), but this resulted in no significant change in posttest colonic temperature (Tc). NE, ISO and EPH produced a preference for a colder region of the gradient (20-22 degrees C) compared to saline (24.5-28.9 degrees C). Posttest Tc was reduced significantly from 37.7-37.9 degrees C after saline to 37.2 degrees C (NE), 37.3 degrees C (ISO), and 36.8 degrees C (EPH). Thus, given the opportunity to select an environmental temperature, the animals selected a Ta that resulted in significantly lower body temperatures after NE, ISO, and EPH. This suggests that paradoxical thermoregulatory effects of these thermogenic adrenergic agonists are due, at least in part, to a preference for a lower body temperature.

This experiment examined whether paradoxical temperature-dependent effects of norepinephrine (NE) can be blocked by the alpha 1-adrenoceptor antagonist WB 4101 (WB) and the Ca(2+)-channel blocker nifedipine. An operant lever-pressing task was used to measure the demand for heat in a cold environment. As noted previously, NE alone (250 micrograms/kg) produced a substantial and significant increase in the demand for heat, and yet post-test colonic temperature (Tc) fell. When tested alone, WB and nifedipine also increased the demand for heat, but this was sufficient to maintain Tc. When combined with NE, WB and nifedipine reduced the demand for heat and the fall in Tc such that there were no differences between the effects of the blockers given alone or with NE. These results indicate that paradoxical thermoregulatory effects of NE in the cold can be antagonized effectively by either an alpha 1-adrenoceptor antagonist or a Ca(2+)-channel blocker.

This experiment examined the effects of intraperitoneal ephedrine (5-25 mg/kg) on operant thermoregulatory behavior of rats in the cold (-8 degrees C), and of 10 mg/kg on metabolic rate at a thermoneutral (22 degrees C) ambient temperature (Ta) and in the cold (5 degrees C). Posttest colonic temperature (Tc) decreased dose-dependently in the behavioral tests, yet the demand for heat varied little with respect to saline except for a significant reduction at the lowest dose tested (i.e., there was no compensation for the reduced Tc induced by ephedrine). Ephedrine had a potent thermogenic effect at a Ta of 22 degrees C, increasing both metabolic rate and Tc. In the cold, ephedrine reduced Tc, but this effect could not be accounted for by a reduction in metabolism, which was not significantly different from saline.

This series of experiments examined whether temperature-dependent effects of the alpha-antagonists prazosin and yohimbine compromised their use as blockers of alpha-adrenergic agonist responses in cold-exposed rats. An operant leverpressing task was used to measure the demand for heat in a cold environment. The alpha 1-antagonist prazosin had modest effects, but the alpha 2-antagonist yohimbine was thermolytic in that it dose dependently increased operant responding but decreased posttest colonic temperature (Tc). These potent effects of the alpha 2-antagonist led to tests of the alpha 2-agonist clonidine. Clonidine increased operant responding for heat to an extraordinary degree, resulting in significant increases in posttest Tc. However, clonidine was found to be a hypothermic agent when tested in rats at 5 degrees C but denied the opportunity to increase body temperature by operant lever pressing, suggesting a central effect on the control of thermal balance. Measurement of changes in metabolic rate at 5 and 23 degrees C showed that yohimbine increased metabolism at 23 degrees C but decreased it in the cold. Prazosin had little effect on metabolism or Tc at either temperature. Prazosin inhibited the decrease in Tc induced by norepinephrine (NE), but had little effect on the lever-pressing response. Yohimbine had no significant antagonistic effect on NE-induced changes in lever-pressing behavior or posttest Tc, but neither did the thermolytic effects of yohimbine exacerbate those of NE. These results show that alpha-antagonist interactions with agonists can be complicated by temperature-dependent effects of each.

This series of experiments examined whether epinephrine (EPI) produces the same thermoregulatory effects in the cold that have been reported for norepinephrine and isoproterenol. Lean and obese Zucker rats were trained to press a lever to activate infrared heat lamps in a cold (-8 degrees C) environment. Operant thermoregulatory behavior increased dose-dependently following EPI (0-100 micrograms/kg), but posttest colonic temperature (Tc) fell. Thermal balance calculations showed a substantial increase in net heat loss, more so in obese than lean animals. EPI is therefore thermolytic--i.e., disrupts thermal balance. A low dose (100 micrograms/kg) of the alpha-antagonist phentolamine produced a marked improvement in operant behavior, Tc, and thermal balance, whereas a comparable dose of the beta-antagonist propranolol had no beneficial effect. Increasing the dose of phentolamine worsened the responses with respect to the 100-micrograms/kg dose. The selective alpha 1-antagonist prazosin ameliorated the decrease in Tc induced by EPI but had little effect on operant behavior or thermal balance; the selective alpha 2-antagonist yohimbine had no effect on any parameter compared to EPI alone. These results suggest that the paradoxical effects of EPI in the cold are mediated by alpha-adrenoceptors, but definitive identification of the subclass of receptor involved cannot be determined.

This experiment examines whether the thermoregulatory ability of obese Zucker rats is comparable to that of lean rats following treatment with beta-adrenoceptor agonists and antagonists in a cold (-8 degrees C) environment. Half-maximal doses of the nonselective beta-adrenoceptor agonist isoproterenol (ISO) produced net thermolytic (heat loss) effects in both obese and lean rats in an operant lever pressing for radiant heat task. ISO increased the demand for heat, but posttest colonic temperature (Tc) decreased. A low dose of propranolol (100 micrograms/kg) normalized thermoregulatory behavior, Tc, and thermal balance when coadministered with ISO. Activation of thermogenesis with the selective beta 3-agonist BRL 35135 (BRL) reduced heat influx by both obese and lean rats at doses between 2 and 10 micrograms/kg, but no dose-response effects were evident within this range. Posttest Tc and thermal balance indicated no thermolytic effects. No evidence was found for a beta 2-component in the BRL response when a supramaximal dose (40 micrograms/kg) was tested with the selective beta 2-antagonist ICI 118551 (1 mg/kg). These data show that, despite a higher baseline demand for heat, the obese Zucker rat responds to the thermogenic effects of BRL and the thermolytic effects of ISO as does the lean rat.

Dose-dependent effects of the selective beta 1 adrenergic antagonist atenolol and the beta 2 antagonist erythro-dl-1-(7-methylinden-4-yloxy)-3-isopropylamino-2-ol were tested on the thermoregulatory responses elicited by half-maximal thermogenic doses of the nonselective beta agonist isoproterenol (ISO) in a cold environment. ISO alone increased operant responding for exogenous heat but decreased body temperature and increased net heat loss. These effects of ISO were blocked by erythro-dl-1-(7-methylinden-4-yloxy)3-isopropylamino-2-ol in a dose-dependent manner, whereas atenolol at the highest dose tested (2 mg/kg) exacerbated the effects of ISO. The beta 3 agonist, (R*,R*-(+/-)-methyl 4-[2-[2-hydroxy-2-(3-chlorophenyl)ethylamino]propylphenoxyacetate hydrobromide, reduced operant responding for heat and net heat loss in the cold; these effects were sustained in the presence of both beta 1 and beta 2 antagonists. The beta 2 adrenoceptor agonist, fenoterol, produced a dose-dependent increase in operant behavior but colonic temperature fell and thermal balance reflected the characteristic effects of a thermolytic agent. The beta 1 agonist, prenalterol, had no apparent effect on thermoregulatory behavior, colonic temperature or thermal balance but it blocked the thermolytic effects of fenoterol when both agonists were coadministered. Fenoterol had no significant effect on metabolic rate or colonic temperature when tested in a warm ambient temperature of 23 degrees C but it decreased colonic temperature without a significant effect on metabolic rate at an ambient temperature of 5 degrees C, suggesting an effect on heat loss mechanisms.(ABSTRACT TRUNCATED AT 250 WORDS)

Four experiments were conducted to examine the effect of ambient temperature (Ta) on norepinephrine (NE)-induced metabolism. NE increased oxygen consumption at ambient temperatures above and at acclimation temperature and decreased oxygen consumption at ambient temperatures below acclimation temperature in both nonacclimated (25 degrees C) and cold-acclimated (5 degrees C) rats. Varying the Ta between -5 degrees and 25 degrees C at a fixed dose of NE (250 micrograms/kg, IP) resulted in a temperature-dependent decrease in metabolism in nonacclimated rats as a function of Ta. A similar effect was demonstrated in cold-acclimated rats tested at -15, 5, and 25 degrees C. Varying the dose of NE between 100 and 1,000 micrograms/kg at a Ta of 25 degrees C resulted in a maximal thermogenic response at a dose of 250 micrograms/kg with diminished responsiveness at higher and lower doses. At 5 degrees C, NE inhibited metabolism maximally at a dose of 250 micrograms/kg. Propranolol, a nonspecific beta-antagonist, attenuated the hypometabolic effect of NE in the cold, while the alpha 2-antagonist yohimbine completely blocked this effect. These results suggest that the metabolic suppressive effect of NE may be mediated by the presynaptic alpha 2-receptor and that beta-adrenoceptors may also contribute to this effect.

The thermoregulatory effects of isothermogenic doses of isoproterenol (Iso) and a novel beta-agonist (BRL 35135) were tested in rats at 22 degrees C and in rats trained to bar press for radiant heat at -8 degrees C. BRL 35135 produced hyperthermia at 22 degrees C and reduced operant responding for heat at -8 degrees C, whereas Iso reduced body temperature and increased operant responding. In both situations, the negative effects of Iso on thermal balance were abolished by propranolol at doses that did not inhibit heat production. In anesthetized rats, propranolol potentiated the Iso-induced rise in brown adipose tissue and colonic temperature. The potentiation was more marked with the beta 2-selective antagonist ICI 118,551, whereas treatment with the beta 1-selective antagonist atenolol resulted in a profound Iso-induced reduction in temperature. The two selective antagonists also produced divergent responses in operant behavior in Iso-treated rats at -8 degrees C. These experiments demonstrate the extent to which responses to a nonselective agonist can be manipulated using appropriately low doses of selective antagonists and indicate that the effects of Iso on thermal balance are due to its beta 2 activity.

The effects of submaximal and maximal thermogenic doses of isoproterenol (ISO) on operant thermoregulatory responses in a cold (-8 degrees C) environment were tested in lean (+/?) Zucker rats trained to barpress for radiant heat. Contrary to expectations, ISO rats pressed for twice as much exogenous heat as controls, but showed a smaller rise in colonic temperature. Conversely, a beta 3-selective adrenergic agonist (RO40-2148) decreased the requirement for exogenous heat and produced larger rises in colonic temperature. RO40-2148 and another beta 3-agonist (ICI D7114) produced similar responses in obese (fa/fa) Zucker rats, but tests with ISO were terminated because it caused profound, and lethal hypothermia. The hypothermic effects of ISO on colonic temperature were also observed in Sprague-Dawley rats at room temperature (22 degrees C), whereas RO40-2148 produced hyperthermia. These results provide behavioral evidence for the high thermogenic selectivity of these novel adrenergic agonists and support the existence of an atypical beta 3-adrenoceptor. The hypothermic effects of ISO are presumed to be due to actions on beta 1- and/or beta 2-adrenoceptors.

The effect of norepinephrine (NE, 250 microgram/kg IP) on thermoregulatory behavior of rats in a cold environment was tested. Norepinephrine produced an increase in operant responding for heat reward but a decrease in core temperature. Since animals compensate behaviorally for alterations in autonomic thermoregulation, this suggested that NE might inhibit cold-induced thermogenesis, an effect contrary to the expected action of this agent. A second experiment showed that NE increased oxygen consumption when rats were tested at 25 degrees C as expected, but decreased oxygen consumption when tested at 5 degrees C. The beta-adrenoceptor antagonist propranolol decreased oxygen consumption both at 25 and at 5 degrees C as expected. These results suggest that the thermogenic effect of NE is highly dependent on ambient temperature.

The effects of a lowered rearing temperature on body weight, core temperature (Tc) and norepinephrine(NE)-stimulated thermogenesis were investigated in 16- to 17-day-old Zucker rat pups. 16-day-old fatty pups were significantly heavier (9%) than lean littermates in litters reared at 18 degrees C ("cold-reared") but not in litters reared at 25 degrees C ("normally-reared"). After 2 h isolation at 25 degrees C, Tc of lean pups was slightly higher (37.1 degrees C) in cold-reared litters than in normally-reared litters (36.4 degrees C), while fatty pups reared at either temperature were severely hypothermic (Tc = 33 - 34 degrees C). At an ambient temperature of 25 degrees C Tc in fatty and lean cold-reared pups increased to 39.5 degrees C after subcutaneous injection of 800 micrograms/kg NE. Normally-reared lean pups reached the same peak Tc after NE injection, while their fatty littermates reached a significantly lower peak Tc of 38.4 degrees C. The hypothermia associated with the onset of excess fat deposition in suckling fatty Zucker rats is not caused by a reduced capacity for NE-stimulated thermogenesis.

Carcass composition was determined in intact and adrenalectomized (ADX) ob/ob mice after 6 wk of food restriction from weaning (21 days). Diets (2.6 or 3.2 g/day) led to reduced weight gain in intact ob/ob mice, but fat deposition was still greater, and lean growth was less than isocalorically fed lean mice. When diet was combined with ADX, obese mice fed 3.2 g/day had rates of fat-free body growth equal to ad libitum-fed lean mice but still gained more weight and deposited more fat. With 2.6 g/day, however, body weight gain, fat deposition, fat-free body growth, and protein deposition were all similar to intact lean mice receiving the same diet. Neither diet nor diet combined with ADX improved the defective thermoregulation of obese mice tested at 23 degrees C, 8 degrees C, or in response to food deprivation or food ingestion. Together, diet and adrenalectomy reduced blood glucose and insulin levels in ob/ob mice, and with the 2.6 g/day diet, the values of each were the same as littermate lean controls. The present results indicate that adrenal glucocorticoids are necessary for maintaining elevated fat deposition at the expense of lean body growth in dieted ob/ob mice, whereas hyperphagia, defective thermoregulation, energy efficiency, and diabetes are still expressed after surgery.

The hypothesis that neuroleptic drugs interfere with operant behaviors by attenuating the rewarding properties of positive reinforcers, was examined in rats trained to lever-press for external heat in a cold environment. Unlike traditional reinforcers, such as food and water, reducing the reward magnitude of heat (by reducing the intensity or duration of the stimulus) results in compensatory increases in operant responding. Neuroleptic pretreatment (0.1, 0.2, 0,4 mg/kg of alpha-flupenthixol) produced only dose-dependent decreases in responding thereby interfering with the animals' ability to behaviorally maintain their internal core temperature. In a temperature-gradient test paradigm (requiring less physical effort on the part of the subjects) alpha-flupenthixol did not alter the animals' preferred environmental temperature, nor did it disrupt behavioral thermoregulatory ability. These data suggest that at least part of the behavioral deficit observed during neuroleptic treatment is due to a disruption in the performance capabilities of the subjects.

The effects of preoptic/anterior hypothalamic injections of acetylcholine on heat-escape behavior and hypothalamic temperature were investigated in unrestrained rats implanted with bilateral cannulae. Two types of responses were observed, either a fall in hypothalamic temperature coupled with an increase in behavioral responses to escape heat or a rise in hypothalamic temperature associated with little or no change in behavioral heat-escape responses. The fall in hypothalamic temperature observed in one group of rats was significant and was associated with a dose-dependent increase in heat-escape responding. The rise in hypothalamic temperature noted in the other group of rats was nonsignificant, and the associated behavioral responses were variable. Distinct anatomical differences in cannulae loci between the 2 groups were not apparent. It is concluded that acetylcholine activates the heat-dissipating control system of the rat hypothalamus and that the hyperthermic effects of acetylcholine are nonspecific.

Rats were trained to work for radiant heat in a cold environment in an apparatus that permitted the simultaneous measurement of metabolic heat production. Reductions in behavioral efficacy produced either by a decrease in reward duration or intensity or by an increase in response effort resulted in a reduction in behavioral heat intake, an increase in metabolic heat production, and no change in body temperature. Thus, autonomic and behavioral temperature regulation interact to produce a constant body temperature.

The body temperatures of mature lean and obese C57BL/6J mice were measured just after feeding, during ad libitum access to food, or every 24 h throughout a 3-day fast. Obese mice had body temperatures 1.0-1.4 degrees C lower than lean mice in the postprandial state and during ad libitum feeding. During food deprivation, obese mice became more hypothermic than lean sex-matched controls. A 5 degrees C fall in body temperature was observed in mutant females in the first 24 h of starvation, about twice that seen in any other experimental group. Over the same period the temperature changes of obese males and lean females were similar and both groups had larger hypothermic responses than lean males. The present results indicate that both genotype and gender affect thermoregulation in these mice. Under normal colony room conditions (ad libitum feeding, 23 degrees C) the ob/ob mutation is expressed by lower body temperatures which along with hypoactivity and hyperphagia account for the high rates of energy storage. When food availability is limited, females of both phenotypes display an increased capacity to reduce their maintenance energy requirements by lowering body temperatures. This hypothermia may be responsible for both the increased conservation of body mass seen during starvation and the slightly greater (5%) fat stores observed in female mice.

Measurements were made of the oxygen consumption and carbon dioxide production of lean and obese Zucker rats ranging from 4 to 400 days of age. As expected, O2 consumption per animal increased with age and was higher in obese than in lean rats. However, O2 consumption was lower in obese animals when expressed per unit of metabolic body mass. Age per se did not influence the respiratory quotient (R), but obese animals had consistently higher Rs than lean animals. These results confirm and extend previous findings described in the literature.

Rats exposed to the cold (-9 degrees C) were trained to depress a lever to obtain pulses of infrared heat. Half of the animals received bilateral electrolytic lesions of the lateral hypothalamus and half were sham operated. Lesioned and control animals performed similarly postoperatively and responded appropriately to changes in reward duration and intensity. Thus, lateral hypothalamic lesions do not impair behavioral thermoregulation. Despite normal behavioral heat intake, body temperature of the lesioned rats fell significantly during the sessions, which can be attributed to an impairment in metabolic heat production due to the lesions.

Plasma levels of total thyroxine (T4) and corticosteroid-binding activity (CBA) were determined in intact, ovariectomized, or ovariectomized, estrogen-treated rats during treatment with propylthiouracil (PTU) and following recovery from PTU. Propylthiouracil treatment reduced the mean plasma T4 concentration to 9 ng/ml and the mean plasma CBA to 39% of normal, while following recovery from PTU, the mean values for plasma T4 and CBA were 56 ng/ml and 108%, respectively. There was a strong (r equals plus +0.89) relationship between plasma levels of T4 and CBA as determined by linear regression analysis. Ovariectomy had no effect on either plasma T4 or CBA in euthyroid animals but significantly decreased CBA in PTU-treated, hypothyroid animals. The results indicate a robust influence of thyroid activity on plasma CBA and suggest a possible interaction between the thyroid and ovary in control of CBA.