Rose Z. Terwilliger

Chronic Cocaine Treatment Decreases Levels of the G Protein Subunits Giα and Goα in Discrete Regions of Rat Brain

Abstract: A possible role for G proteins in contributing to the chronic actions of cocaine was investigated in three rat brain regions known to exhibit electrophysiological responses to chronic cocaine: the ventral tegmental area, nucleus accumbens, and locus coeruleus. It was found that chronic, but not acute, treatment of rats with cocaine produced a small (∼ 15%), but statistically significant, decrease in levels of pertussis toxin‐mediated ADP‐ribosylation of Giα and Goα in each of these three brain regions. The decreased ADP‐ribosylation levels of the G protein subunits were shown to be associated with 20–30% decreases in levels of their immunoreactivity. In contrast, chronic cocaine had no effect on levels of G protein ADP‐ribosylation or immunoreactivity in other brain regions studied for comparison. Chronic cocaine also had no effect on levels of GSα or Gβ immunoreactivity in the ventral tegmental area and nucleus accumbens. Specific decreases in Giα and Goα levels observed in response to chronic cocaine in the ventral tegmental area, nucleus accumbens, and locus coeruleus are consistent with the known electrophysiological actions of chronic cocaine on these neurons, raising the possibility that regulation of G proteins represents part of the biochemical changes that underlie chronic cocaine action in these brain regions.

Regulation of G proteins by chronic morphine in the rat locus coeruleus

A possible role for G proteins in contributing to the chronic actions of opiates was investigated in the rat locus coeruleus (LC). The LC is a relatively homogeneous brain region that appears to play an important role in mediating acute and chronic opiate action in animals, as well as in humans. It was found that chronic, but not acute, treatment of rats with morphine, under conditions known to induce states of opiate tolerance and dependence, produced an increase in the level of pertussis toxin-mediated ADP-ribosylation of G proteins in the LC. The morphine-induced increase in ADP-ribosylation occurred in both Gi and Go, and was observed over a 30-fold range of NAD concentrations used. Concomitant treatment of rats with the opiate receptor antagonist naltrexone blocked the ability of morphine to produce this effect. In contrast, chronic morphine had no effect on pertussis toxin-mediated ADP-ribosylation of Gi and Go in the other brain regions studied, including the neostriatum, frontal cortex, and dorsal raphe. Chronic morphine also had no effect on cholera toxin-mediated ADP-ribosylation of Gs in the LC and these other brain regions. Preliminary immunoblot analysis revealed that increased ADP-ribosylation levels of the alpha subunit of Go in the LC were associated with equivalent increases in the immunoreactivity of this protein in this brain region. It is possible that the observed regulation of G-proteins by morphine in the LC represents part of the changes that underlie opiate addiction in these neurons.

Chronic antidepressant administration alters the subcellular distribution of cyclic AMP-dependent protein kinase in rat frontal cortex.

Abstract: The influence of chronic administration of antidepressants on cyclic AMP‐dependent protein kinase activity was examined in rat frontal cortex. Chronic administration of imipramine, tranylcypromine, or electroconvulsive seizures decreased cyclic AMP‐dependent protein kinase activity in soluble fractions by ∼25%, whereas enzyme activity was increased in the particulate fractions by ∼20%. In contrast, enzyme activity in crude homogenates was not altered. This effect appears to be specific to antidepressant jdrugs, because representatives of several other classes of psychotropic drugs—namely, haloperidol, morphine, and diazepam—failed to alter either soluble or particulate levels of cyclic AMP‐dependent protein kinase activity in this brain region following chronic administration. When the total particulate fraction was subfractionated, it was found that chronic imipramine treatment significantly increased the activity of cyclic AMP‐dependent protein kinase in crude nuclear fractions but not in crude synaptosomal or microsomal fractions. Taken together, the data raise the possibility that chronic antidepressant treatments may stimulate the translocation of cyclic AMP‐dependent protein kinase from the cytosol to the nucleus. This effect would represent a novel action of antidepressants that could contribute to the long‐term adaptive changes in brain thought to be essential for the blinical actions of these treatments.

Sweet taste signaling functions as a hypothalamic glucose sensor.

Brain glucosensing is essential for normal body glucose homeostasis and neuronal function. However, the exact signaling mechanisms involved in the neuronal sensing of extracellular glucose levels remain poorly understood. Of particular interest is the identification of candidate membrane molecular sensors that would allow neurons to change firing rates independently of intracellular glucose metabolism. Here we describe for the first time the expression of the taste receptor genes Tas1r1, Tas1r2 and Tas1r3, and their associated G-protein genes, in the mammalian brain. Neuronal expression of taste genes was detected in different nutrient-sensing forebrain regions, including the paraventricular and arcuate nuclei of the hypothalamus, the CA fields and dentate gyrus of the hippocampus, the habenula, and cortex. Expression was also observed in the intra-ventricular epithelial cells of the choroid plexus. These same regions were found to express the corresponding gene products that form the heterodimeric T1R2/T1R3 and T1R1/T1R3 sweet and l-amino acid taste G-protein coupled receptors, respectively, along with the taste G-protein α-gustducin. Moreover, in vivo studies in mice demonstrated that the hypothalamic expression of taste-related genes is regulated by the nutritional state of the animal, with food deprivation significantly increasing expression levels of Tas1r1 and Tas1r2 in hypothalamus, but not in cortex. Furthermore, exposing mouse hypothalamic cells to a low-glucose medium, while maintaining normal l-amino acid concentrations, specifically resulted in higher expression levels of the sweet-associated gene Tas1r2. This latter effect was reversed by adding the non-metabolizable artificial sweetener sucralose to the low-glucose medium, indicating that taste-like signaling in hypothalamic neurons does not require intracellular glucose oxidation. Taken together, our findings suggest that the heterodimeric G-protein coupled sweet receptor T1R2/T1R3 is a candidate membrane-bound brain glucosensor.

Chronic Antidepressant Administration Increases the Expression of cAMP-Specific Phosphodiesterase 4A and 4B Isoforms

The influence of chronic antidepressant administration on expression of the three major phosphodiesterase (PDE) 4 subtypes found in brain (PDE4A, PDE4B, and PDE4D) was examined. The treatments tested included representatives of four major classes of antidepressants: selective reuptake inhibitors of serotonin (sertraline and fluoxetine) or norepinephrine (desipramine), a monoamine oxidase inhibitor (tranylcypromine), and electroconvulsive seizure. Expression of PDE4A and PDE4B, but not PDE4D, mRNA and immunoreactivity were significantly increased in rat frontal cortex by chronic administration of each of the four classes of antidepressants. We also found that antidepressant administration significantly increased the expression of PDE4B mRNA in the nucleus accumbens, a brain region thought to mediate pleasure and reward that could also contribute to the anhedonia often observed in depressed patients. In contrast, expression of PDE4A and PDE4B were not influenced by short-term treatment (1 or 7 d) and were not influenced by chronic administration of nonantidepressant psychotropic drugs (cocaine or haloperidol), demonstrating the time dependence and pharmacological specificity of these effects. Upregulation of PDE4A and PDE4B may represent a compensatory response to antidepressant treatment and activation of the cAMP system. The possibility that targeted inhibition of these PDE4 subtypes may produce an antidepressant effect is discussed.

Biochemical Adaptations in the Mesolimbic Dopamine System in Response to Repeated Stress

We have demonstrated previously that chronic administration of morphine, cocaine, or ethanol produces some common biochemical adaptations in the ventral tegmental area (VTA) and nucleus accumbens (NAc), components of the mesolimbic dopamine system implicated in the reinforcing and locomotor activating properties of these drugs of abuse. Because this neural pathway is also regulated by stress, and because stress has been shown to influence an animals behavioral responses to drugs of abuse, it was of interest to determine whether repeated exposure to stress results in similar biochemical adaptations. By use of immunoblot analysis, we show here that a course of chronic “unpredictable” stress, like chronic drug exposure, increased levels of immunoreactivity of tyrosine hydroxylase and glial fibrillary acidic protein and decreased levels of immunoreactivity of neurofilament proteins in the VTA. Chronic unpredictable stress also increased levels of cyclic AMP-dependent protein kinase activity and decreased levels of immunoreactivity of the G protein subunit, Giα, in the NAc. These effects required long-term exposure to stress and were in most cases not seen in the substantia nigra and caudate-putamen, components of the nigrostriatal dopamine system studied for comparison. The biochemical effects of chronic stress in the VTA and NAc differed among three strains of rat studied. Fischer 344 rats were the most responsive in that they exhibited all of the aforementioned adaptations, whereas Lewis rats were the least responsive in that they exhibited none of these adaptations; Sprague-Dawley rats exhibited an intermediate number of responses. Taken together, the results of the present study demonstrate that chronic exposure to stress results in biochemical adaptations in the mesolimbic dopamine system that resemble the chronic actions of several drugs of abuse. These adaptations could contribute to the convergent behavioral effects induced by treatments that are mediated via the VTA-NAc pathway.

Coordinate regulation of the cyclic AMP system with firing rate and expression of tyrosine hydroxylase in the rat locus coeruleus: effects of chronic stress and drug treatments.

Abstract: Recent studies have demonstrated that chronic stress increases the firing rate and expression of tyrosine hydroxylase (TH) in neurons of the locus coeruleus (LC), the major noradrenergic nucleus in brain. The present study was undertaken to examine the influence of chronic stress and other treatments known to influence the activity of LC neurons on the cyclic AMP (cAMP) second messenger system in these neurons. Chronic (5 days) cold exposure significantly increased levels of TH immunoreactivity in the LC, as previously reported, but not in substantia nigra (SN) or ventral tegmentum (VT), two dopaminergic nuclei studied for comparison. Chronic cold exposure increased levels of cAMP‐dependent protein kinase activity in soluble, but not particulate, fractions of the LC, and increased basal and GTP‐ and forskolin‐stimulated adenylate cyclase activity in this brain region. In contrast, levels of the protein kinase and adenylate cyclase in VT, SN, and frontal cortex were not significantly influenced by cold exposure. To study further the relationship between regulation of LC firing rate, TH expression, and the cAMP system in the LC, other treatments known to influence TH were examined. Reserpine treatment, shown previously to increase levels of TH, was found to increase both LC firing rate and levels of soluble cAMP‐dependent protein kinase activity in the LC. 6‐Hydroxydopamine, shown previously to increase levels of TH and firing rate of LC neurons, also increased soluble levels of protein kinase activity. Other treatments known to either increase (adrenalectomy) or decrease (chronic imipramine) levels of TH in the LC were also found to increase or decrease, respectively, levels of cAMP‐dependent protein kinase activity in this brain region. The results demonstrate the coordinate regulation of LC firing rate, TH expression, and the cAMP system by chronic stress, catecholamine depletion, and various drug and hormone treatments and raise the possibility that adaptations in the cAMP system in response to these treatments contribute to regulation of LC neuronal firing and TH expression.

Role of 5-HT2A receptors in the stress-induced down-regulation of brain-derived neurotrophic factor expression in rat hippocampus

Immobilization stress decreases the expression of BDNF mRNA in the rat hippocampus, and this effect could contribute to the atrophy of hippocampal neurons. This study examines the influence of selective 5-HT, as well as norepinephrine, receptor antagonists on the stress-induced down-regulation of BDNF mRNA. Pretreatment with a selective 5-HT2A receptor antagonist, MDL100,907, significantly blocked the influence of stress on expression of BDNF mRNA. In contrast, pretreatment with either a selective 5-HT2C or 5-HT1A receptor antagonist did not influence the stress-induced decrease in levels of BDNF mRNA. The stress-induced decrease was also not influenced by pretreatment with antagonists of beta(1/2)- or alpha1-adrenergic, or CRF-R1 receptors. The results demonstrate that 5-HT2A receptors mediate, at least in part, the stress-induced down-regulation of BDNF expression in the rat hippocampus.

Lewis and Fischer rat strains display differences in biochemical, electrophysiological and behavioral parameters: studies in the nucleus accumbens and locus coeruleus of drug naive and morphine-treated animals

In previous studies, we demonstrated that tyrosine hydroxylase and neurofilament proteins are regulated by chronic morphine and chronic cocaine treatments in the ventral tegmental area in Sprague-Dawley rats and that the inbred Lewis and Fischer 344 rat strains, under drug-naive conditions, show different levels of these proteins specifically in this brain region. In the current study, we compared Lewis and Fischer rats with respect to levels of adenylate cyclase, cyclic AMP-dependent protein kinase and G-proteins in the nucleus accumbens (NAc) and locus coeruleus (LC), brain regions in Sprague-Dawley rats where these proteins are regulated by chronic exposure to morphine or to cocaine. We found that levels of adenylate cyclase and cyclic AMP-dependent protein kinase activity are higher in the NAc and LC of Lewis rats compared to Fischer rats, whereas levels of Gi alpha and G beta were lower. These strain differences were not seen in several other brain regions analyzed and no strain differences were detected in levels of other G-protein subunits. Lewis and Fischer rats also differed in the ability of chronic morphine to regulate adenylate cyclase and cyclic AMP-dependent protein kinase in the NAc and LC. In the NAc, chronic morphine increased levels of the two enzymes in the Fischer strain only, whereas in the LC chronic morphine increased levels of the enzymes in both strains, with more robust effects seen in the Lewis rat. To understand possible physiological consequences of these strain differences in the cyclic AMP pathway, we studied LC neuronal activity under basal and chronic morphine-treated conditions. LC neurons of Lewis rats showed higher spontaneous firing rates in brain slices in vitro than those of Fischer rats and also showed greater morphine-induced increases in responsiveness to bath-applied 8-bromo-cyclic AMP. These electrophysiological findings are generally consistent with the biochemical observations. Moreover, Lewis and Fischer rats displayed very different opiate withdrawal syndromes, with different types of behaviors elicited upon precipitation of opiate withdrawal with the opiate receptor antagonist, naltrexone. The possible relationship between these behavioral findings and the biochemical and electrophysiological data is discussed. These studies provide further support for the possibility that Lewis and Fischer rat strains provide a useful model system in which some of the genetic factors that contribute to drug-related behaviors can be investigated.

Scopolamine rapidly increases mammalian target of rapamycin complex 1 signaling, synaptogenesis, and antidepressant behavioral responses.

BACKGROUND Clinical studies report that scopolamine, an acetylcholine muscarinic receptor antagonist, produces rapid antidepressant effects in depressed patients, but the mechanisms underlying the therapeutic response have not been determined. The present study examines the role of the mammalian target of rapamycin complex 1 (mTORC1) and synaptogenesis, which have been implicated in the rapid actions of N-methyl-D-aspartate receptor antagonists. METHODS The influence of scopolamine on mTORC1 signaling was determined by analysis of the phosphorylated and activated forms of mTORC1 signaling proteins in the prefrontal cortex (PFC). The numbers and function of spine synapses were analyzed by whole cell patch clamp recording and two-photon image analysis of PFC neurons. The actions of scopolamine were examined in the forced swim test in the absence or presence of selective mTORC1 and glutamate receptor inhibitors. RESULTS The results demonstrate that a single, low dose of scopolamine rapidly increases mTORC1 signaling and the number and function of spine synapses in layer V pyramidal neurons in the PFC. Scopolamine administration also produces an antidepressant response in the forced swim test that is blocked by pretreatment with the mTORC1 inhibitor or by a glutamate alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor antagonist. CONCLUSIONS Taken together, the results demonstrate that the antidepressant actions of scopolamine require mTORC1 signaling and are associated with increased glutamate transmission, and synaptogenesis, similar to N-methyl-D-aspartate receptor antagonists. These findings provide novel targets for safer and more efficacious rapid-acting antidepressant agents.