Induction of innate immune genes in brain create the neurobiology of addiction.

Addiction occurs through repeated abuse of drugs that progressively reduce behavioral control and cognitive flexibility while increasing limbic negative emotion. Recent discoveries indicate neuroimmune signaling underlies addiction and co-morbid depression. Low threshold microglia undergo progressive stages of innate immune activation involving astrocytes and neurons with repeated drug abuse, stress, and/or cell damage signals. Increased brain NF-κB transcription of proinflammatory chemokines, cytokines, oxidases, proteases, TLR and other genes create loops amplifying NF-κB transcription and innate immune target gene expression. Human post-mortem alcoholic brain has increased NF-κB and NF-κB target gene message, increased microglial markers and chemokine-MCP1. Polymorphisms of human NF-κB1 and other innate immune genes contribute to genetic risk for alcoholism. Animal transgenic and genetic studies link NF-κB innate immune gene expression to alcohol drinking. Human drug addicts show deficits in behavioral flexibility modeled pre-clinically using reversal learning. Binge alcohol, chronic cocaine, and lesions link addiction neurobiology to frontal cortex, neuroimmune signaling and loss of behavioral flexibility. Addiction also involves increasing limbic negative emotion and depression-like behavior that is reflected in hippocampal neurogenesis. Innate immune activation parallels loss of neurogenesis and increased depression-like behavior. Protection against loss of neurogenesis and negative affect by anti-oxidant, anti-inflammatory, anti-depressant, opiate antagonist and abstinence from ethanol dependence link limbic affect to changes in innate immune signaling. The hypothesis that innate immune gene induction underlies addiction and affective disorders creates new targets for therapy.

Presynaptic alpha-receptor subsensitivity after long-term antidepressant treatment.

After 3 weeks of twice-daily administration of desipramine to rats, the frequency-response curve for field stimulation of adrenergic neurons in isolated left atrial strips was shifted markedly to the left and the efflux of [3H]norepinephrine was enhanced greatly. After 1 day of treatment, only slight shifts in the frequency-response curve and small increases in [3H]norepinephrine efflux occurred although inhibition of [3H]norepinephrine uptake was already maximal, and phenoxybenzamine caused a further shift to the left in the frequency-response curve similar to that which occurred after 3 weeks of desipramine treatment alone. A gradual decrease in the sensitivity of the presynaptic alpha receptor would explain the delay in the onset of the linical effect of the tricyclic antidepressants.

Distinct cell proliferation events during abstinence after alcohol dependence: microglia proliferation precedes neurogenesis.

Excessive alcohol intake characteristic of Alcohol Use Disorders (AUDs) produces neurodegeneration that may recover with abstinence. The mechanism of regeneration is unclear, however neurogenesis from neural stem/progenitor cells is a feasible mechanism of structural plasticity. Therefore, a timecourse of cell proliferation was examined in a rat model of an AUD and showed a striking burst in cell proliferation at 2 days of abstinence preceding the previously reported neurogenic proliferation at 7 days. New cells at 2 days, assessed by bromo-deoxy-uridine incorporation and endogenous markers, were observed throughout hippocampus and cortex. Although the majority of these new cells did not become neurons, neurogenesis was not altered at this specific time point. These new cells expressed a microglia-specific marker, Iba-1, and survived at least 2 months. This first report of microglia proliferation in a model of an AUD suggests that microgliosis could contribute to volume recovery in non-neurogenic regions during abstinence.

Neurogenesis in adolescent brain is potently inhibited by ethanol.

Adolescence is a period of progressive changes in brain that likely contribute to the maturation of behavior. Human adolescents consume large amounts of ethanol. To investigate the effects of ethanol on adolescent neural progenitor cells, male rats (35-40 days old) were treated with an acute dose of ethanol (1.0, 2.5 or 5.0 g/kg, i.g.) or vehicle that resulted in peak blood levels of 33, 72, and 131 mg/dl, respectively. Bromodeoxyuridine (300 mg/kg i.p.) was administered to label dividing cells and rats were killed at 5 h to assess proliferation or at 28 days to assess cell survival and differentiation. After 5 h, bromodeoxyuridine-immunoreactivity was reduced by 63, 97 and 99% in the rostral migratory stream and 34, 71 and 99% in the subventricular zone by 1.0, 2.5 and 5.0 g/kg of ethanol respectively. In the dentate gyrus, ethanol reduced bromodeoxyuridine-immunoreactivity by 29, 40, and 78% at the three doses respectively. The density of doublecortin immunoreactivity was decreased after 3 days and the number of bromodeoxyuridine+ cells remained decreased at 28 days when most hippocampal bromodeoxyuridine+ cells coexpressed neuronal nuclei, a neuronal marker. These studies indicate that the adolescent brain is very sensitive to acute ethanol inhibition of neurogenesis.

Activation of phosphatidylinositol 3-kinase and phosphatidylinositol 4-kinase during rat parotid acinar cell proliferation.

We have recently shown that beta-adrenergic agonist, isoproterenol-induced parotid acinar cell proliferation is in part mediated by elevated levels of surface galactosyltransferase which undergoes interaction with the EGF-R. The receptor subsequently undergoes autophosphorylation on the tyrosine residues in a manner similar to its 'receptor-ligand' interaction (Purushotham et al. (1992) Biochem. J. 284, 767-776). In this study, we provide evidence for phosphatidylinositol 3-kinase and 4-kinase as cytoplasmic signalling proteins involved in both the isoproterenol and EGF-stimulated signal transduction upon in vitro and in-vivo stimulation of parotid acinar cells. Total cell lysate activity for the PtdIns 4-kinase was 2- and 3-fold higher than unstimulated control cells, while the PtdIns 3-kinase was 1.4- and 2.8-fold higher following stimulation by isoproterenol or EGF, respectively. Increases of 6- and 2-fold in phosphatidylinositol 3-kinase were observed in anti-phosphotyrosine-antibody-immunoprecipitated cell lysates upon in-vitro growth stimulation with isoproterenol or EGF, respectively. There was an increase in tyrosine phosphorylation of the holoenzyme and association of the p85 subunit of phosphatidylinositol 3-kinase with EGF-R in response to both isoproterenol and EGF treatments. This corresponded with the mobilization of p85 from the cytoplasm to the plasma membrane upon growth stimulation. These results further implicate the phosphoinositide metabolites in the second messenger signalling pathways of isoproterenol-induced rat parotid cell proliferation. The parallel utilization of EGF indicate that the post-transductional mechanisms of isoproterenol-induced acinar cell proliferation are similar to the growth-factor-mediated activation of intracellular signalling pathways for cell growth.

Ethanol tolerance and synaptic plasticity.

Current concepts of the mechanisms underlying many of the pharmacological effects of ethanol on the CNS involve disruption of ion channel function via the interaction of ethanol with specific hydrophobic sites on channel subunit proteins. Of particular clinical importance is the development of tolerance and dependence to ethanol, and it is likely that adaptive changes in synaptic function in response to ethanol's actions on ion channels play a role in this process. In this article, Judson Chandler, Adron Harris and Fulton Crews discuss potential mechanisms of ethanol-induced changes in synaptic function that might provide a cellular basis for ethanol tolerance and dependence. It is proposed that multiple mechanisms are involved that include both transcriptional and post-translational modifications in NMDA and GABAA receptors.

Adolescent intermittent ethanol exposure enhances ethanol activation of the nucleus accumbens while blunting the prefrontal cortex responses in adult rat.

The brain continues to develop through adolescence when excessive alcohol consumption is prevalent in humans. We hypothesized that binge drinking doses of ethanol during adolescence will cause changes in brain ethanol responses that persist into adulthood. To test this hypothesis Wistar rats were treated with an adolescent intermittent ethanol (AIE; 5 g/kg, i.g. 2 days on-2 days off; P25-P54) model of underage drinking followed by 25 days of abstinence during maturation to young adulthood (P80). Using markers of neuronal activation c-Fos, EGR1, and phophorylated extracellar signal regulated kinase (pERK1/2), adult responses to a moderate and binge drinking ethanol challenge, e.g., 2 or 4 g/kg, were determined. Adult rats showed dose dependent increases in neuronal activation markers in multiple brain regions during ethanol challenge. Brain regional responses correlated are consistent with anatomical connections. AIE led to marked decreases in adult ethanol PFC (prefrontal cortex) and blunted responses in the amygdala. Binge drinking doses led to the nucleus accumbens (NAc) activation that correlated with the ventral tegmental area (VTA) activation. In contrast to other brain regions, AIE enhanced the adult NAc response to binge drinking doses. These studies suggest that adolescent alcohol exposure causes long-lasting changes in brain responses to alcohol that persist into adulthood.

Ethanol enhances the endothelial nitric oxide synthase response to agonists.

Chronic ethanol consumption is associated with an increased prevalence of hypertension. The mechanisms of this form of hypertension are unknown. Rats fed ethanol for 2 days develop a tolerance to the acute vasoconstrictive effects of ethanol that is believed to be endothelium dependent. We investigated the effects of acute and chronic ethanol exposure on agonist-stimulated nitric oxide synthase activity in bovine pulmonary artery endothelial cells. Exposure of bovine pulmonary artery endothelial cells to ethanol (100 mmol/L) for 20-120 minutes did not change either basal or agonist-stimulated nitric oxide synthase activity measured as the rate of conversion of [3H]L-arginine to [3H]L-citrulline. Chronic exposure of endothelial cells to ethanol (100 mmol/L) for 96 hours significantly increased bradykinin-, adenosine 5'-triphosphate-, and ionomycin-stimulated nitric oxide synthase activity without affecting basal enzyme activity. The ethanol-induced increase in nitric oxide synthase response to agonists was dependent on the duration of ethanol exposure as well as the concentration of ethanol. Moreover, the effect of ethanol was characterized by an increase in the maximal nitric oxide synthase response to adenosine 5'-triphosphate without changes in the EC50. Removal of calcium or addition of N omega-nitro-L-arginine completely abolished agonist-stimulated nitric oxide synthase activity in both control and ethanol-treated cells. Our observations support the hypothesis that ethanol enhances nitric oxide synthase response to agonists during early ethanol exposure and may serve in a protective role against its hypertensive effect.

Decreased carbachol-stimulated inositol 1,3,4,5-tetrakisphosphate formation in senescent rat cerebral cortical slices.

It is well established that muscarinic cholinergic receptors are linked to phosphoinositide hydrolysis in brain. Previous studies of muscarinic responses used Li+ to increase inositol phosphate accumulation and suggested little or no change during aging. Li+ disrupts certain aspects of the inositol phosphate metabolism and inhibits the formation of inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P4]. Ins(1,3,4,5)P4 appears to have second messenger functions. To investigate the effects of aging on agonist stimulated Ins(1,3,4,5)P4 formation, young (6-8 months) and old (28-30 months) Fischer 344 rat cerebral cortical or hippocampal slices were challenged with various agonists known to stimulate phosphoinositide hydrolysis in brain using a recently developed assay that does not use Li+. Carbachol and quisqualate stimulated [3H]inositol trisphosphate ([3H]InsP3) and [3H]Ins(1,3,4,5)P4 formation in young and old rat cerebral cortical slices. Norepinephrine, 5-hydroxytryptamine, and vasopressin failed to stimulate [3H]Ins(1,3,4,5)P4 or [3H]InsP3 formation in either young or old rat cerebral cortical slices. In old rat cerebral cortical slices, the carbachol-stimulated [3H]Ins(1,3,4,5)P4 formation was reduced by 44%. Angiotensin II stimulated [3H]InsP3 was increased (219%) in old rats. There was no influence of aging either on the basal level or on the maximal response to carbachol or quisqualate in hippocampal slices. These studies suggest region-specific changes in phosphoinositide hydrolysis during aging.

Effects of nBM lesions on muscarinic-stimulation of phosphoinositide hydrolysis.

The nucleus basalis magnocellularis (nBM) is believed to be the major path of cholinergic innervation to the frontal cortex. The cerebral cortex is known to contain muscarinic receptors that are coupled to the hydrolysis of phosphoinositides (PI) (9,14). Adult male Sprague-Dawley rats were unilaterally and bilaterally lesioned at the nBM with the excitotoxin ibotenic acid and killed at 7 or 21 to 23 days postsurgery. In rats unilaterally lesioned 7 days previously, the carbachol dose-response curves in lesioned fronto-parietal cortex were identical to control fronto-parietal cortices. In rats studied 21 to 23 days postsurgery, carbachol dose-response curves were again identical in control vs. lesioned fronto-parietal cortices. Similar results are obtained when bilaterally lesioned rats are compared to sham-operated controls. For each group, the hydrolysis is linear with respect to time until 15 minutes with a maximum reached at approximately 40 minutes. Receptor density, as measured by [3H]-QNB binding or agonist competition for [3H]-QNB binding, was not changed by any of the lesions studied. These results suggest that the loss of cholinergic innervation from the nBM does not result in compensatory denervation supersensitivity in cerebral fronto-parietal cortical muscarinic receptors.

Effects of aging on rat cortical presynaptic cholinergic processes.

We studied the effects of aging on the [3H]-choline uptake, acetylation, [3H]-ACh release and muscarinic modulation of [3H]-ACh release in cortical synaptosomes prepared from Fischer 344 male rats. Our results indicate that 6 and 24 month old rats take up and acetylate [3H]-choline to a similar extent, but that the older animals release significantly less [3H]-ACh in response to K+-depolarization than the young adults do. This difference in K+-induced release is not due to a difference in presynaptic muscarinic receptor inhibitory activity since the older animals appear to be, if anything, slightly less sensitive to oxotremorine than the younger animals are. Atropine (1 microM) had no effect on ACh-release but blocked oxotremorine-induced modulation. Our results suggest that acetylcholine release is decreased in synaptosomes prepared from old rats although the presynaptic muscarinic regulation of release is functional. Thus, muscarinic receptor-mediated release-modulation is a potential site for pharmacologically altering ACh release.

Down-regulation of serotonin2, but not of beta-adrenergic receptors during chronic treatment with amitriptyline is independent of stimulation of serotonin2 and beta-adrenergic receptors.

Antidepressant drugs down-regulate beta-adrenergic, alpha 2-adrenergic and serotonergic 5-HT2 receptors with a time course that parallels their clinical efficacy, i.e. chronic administration is required (Crews and Smith, 1978; Svensson and Usdin, 1978; Banerjee, Kung, Riggi and Chanda, 1979; Bergstrom and Keller, 1979; Peroutka and Snyder, 1980). In the present study, it was found that the 5-HT2 receptor antagonist, nefazadone (50 mg/kg per day) did not prevent the downregulation of 5-HT2 receptors in the cerebral cortex produced by amitriptyline (10 mg/kg per day), when administered for 3 weeks. Moreover, treatment with nefazadone (50 mg/kg per day) alone for 3 weeks decreased binding to 5-HT2 receptors in cerebral cortex. In contrast, administration of propranolol, the beta receptor antagonist, (10 mg/kg per day) with amitriptyline (10 mg/kg per day) for 3 weeks prevented the down-regulation of beta receptors, but did not alter the decrease in binding to 5-HT2 receptors. In addition, the depletion of central stores of norepinephrine and serotonin by a 4-day treatment with reserpine (5 mg/kg per day) increased binding to beta receptors in the cerebral cortex and hippocampus, but did not affect binding to 5-HT2 receptors in either region. These results suggest that the 5-HT2 receptor is not down-regulated by direct stimulation by serotonin agonists and that the down-regulation of 5-HT2 receptors by amitriptyline is independent of down-regulation of beta-adrenergic receptors.

Differences in imidazoline and phenylethylamine alpha-adrenergic agonists: comparison of binding affinity and phosphoinositide response.

The imidazoline class of compounds, reported to be partial agonists at alpha 1 adrenoceptors, were compared with phenylethylamines for their ability to displace the binding of [3H]prazosin and to stimulate hydrolysis of phosphoinositides in the cerebral cortex of the rat. Both classes of alpha adrenoceptor compounds exhibited two sites of interaction with binding sites for [3H]prazosin in 30 mM Tris buffer. In a Na+ containing ionic buffer, the competition by phenylethylamines for [3H]prazosin sites shifted to a one-site best-fit, while imidazolines retained their two-site best-fit. Phenylethylamines stimulated hydrolysis of phosphoinositides in a dose-dependent manner, with ED50 values that correlated with Kd values from competition curves. In contrast, imidazolines were not potent or efficacious at stimulating hydrolysis of phosphoinositides and the binding affinities did not correlate with the ED50 values. The alpha 1 adrenoceptor antagonist, prazosin potently inhibited phenylethylamine, but not imidazoline-stimulated hydrolysis of phosphoinositides. Dose-response curves to the imidazoline, oxymetazoline, in the presence and absence of maximally stimulating concentrations of norepinephrine, indicated that oxymetazoline caused a dose-dependent inhibition of norepinephrine-stimulated hydrolysis of phosphoinositide. The inhibition of norepinephrine-stimulated hydrolysis of phosphoinositides was evident up to 100 microM, at which point oxymetazoline elicited hydrolysis of phosphoinositides through a non-alpha 1 adrenoceptor-mediated mechanism. These data indicate that imidazolines act primarily as antagonists at the alpha 1 adrenoceptor, coupled to hydrolysis of phosphoinositide and stimulate the hydrolysis of phosphoinositide through a non-alpha 1 adrenoceptor mechanism.

Rapid down-regulation of serotonin2 receptor binding during combined administration of tricyclic antidepressant drugs and alpha 2 antagonists.

Recent studies have shown that chronic, but not acute, treatment with several different antidepressant drugs decreases the density of serotonin2 (5HT2) receptors in the cerebral cortex (Peroutka and Snyder, 1980a). It was found that combined administration of antidepressant drugs with alpha 2 antagonists could accelerate this down-regulation of 5HT2 receptors induced by antidepressants. Amitriptyline (10 mg/kg, twice daily) in combination with yohimbine (5 mg/kg, twice daily) decreased 5HT2 receptor binding after 4 days of treatment, whereas amitriptyline alone required 10 days to decrease the density of 5HT2 receptors. Other antidepressant--alpha-antagonist combinations which rapidly decreased the density of 5HT2 receptors were desipramine-phenoxybenzamine, amitriptyline-phenoxybenzamine, iprindole-phenoxybenzamine, mianserin-phenoxybenzamine and desipramine-dihydroergotamine. Treatment with desipramine in combination with prazosin, an alpha 1 antagonist, did not accelerate the down-regulation of 5HT2 receptors induced by antidepressants. These results suggest that blockade of central alpha 2 receptors can accelerate the down-regulation of 5HT2 receptors by antidepressants. A combination of antidepressant and alpha antagonist treatment could represent a new, rapid onset antidepressant drug therapy.

Receptors, phosphoinositol hydrolysis and plasticity of nerve cells.

Excitatory amino acid neurotransmission has been shown to be necessary but may not be sufficient, for the production of LTP and other prolonged changes in synaptic transmission. Excitatory neurotransmission may produce depolarization-induced increases in intracellular calcium that cause PI hydrolysis and synergistically potentiate receptor-G protein induced PI hydrolysis. This synergistic potentiation of phosphoinositide hydrolysis, and increased [Ca]i due to positive cross stimulation, may lead to depolarization block, a persistent increase in protein kinase activation, altered morphology, oncogene activity and other plasticity changes important in memory.

Changes in cortical synaptosomal plasma membrane fluidity and composition in ethanol-dependent rats.

Synaptosomal plasma membranes (SPM) were examined from the following four groups of rats: controls; rats acutely treated with single doses of ethanol; a prodromal-detoxication group (dependent-intoxicated); rats undergoing overt ethanol-withdrawal syndrome. Estimates of the apparent microviscosity of SPM over a range of temperatures indicated that temperature-induced changes in SPM fluidity were smaller during the prodromal detoxication phase. The cholesterol:phospholipid molar ratio significantly increased in SPM from the prodromal-phase rats, but to a lesser extent in rats undergoing ethanol withdrawal syndrome. The fatty acid content of SPM phospholipids was not significantly changed in any of the treatment groups. Addition of cholesterol in vitro to control membranes altered the apparent microviscosity similarly to the changes found in SPM of ethanol-dependent rats. These studies suggest that physical dependence upon ethanol may be related to changes in synaptosomal membrane composition and viscosity.

Uncoupling of muscarinic cholinergic phosphoinositide signals in senescent cerebral cortical and hippocampal membranes.

Muscarinic-cholinergic signals in brain are mediated in part through the hydrolysis of phosphoinositides (PtdIns) by phospholipase C (PLC). To test the hypothesis that muscarinic PtdIns signals change during aging, membranes were prepared from the cerebral cortex and hippocampus of young (4-6 months old), middle aged (8-10 months old) and senescent (24-26 months old) Fisher 344 rats. Carbachol dose-dependently increased [3H]-PtdIns hydrolysis in both brain regions in all three age groups, however, in senescent rats the maximal response was decreased to 69.26 +/- 4.33% (p < 0.01) in cortex and to 48.29 +/- 2.55% (p < 0.01) in hippocampus of young rat values. In contrast to the decrease in carbachol-stimulated phosphoinositide hydrolysis, calcium-stimulated phosphoinositide hydrolysis was not altered. GTP gamma S also dose-dependently increased [3H]-PtdIns hydrolysis in membranes from all three age groups through G-protein-PLC activation. Similar to carbachol, GTP gamma S-activated [3H]-PtdIns hydrolysis was reduced approximately 40% in senescent rats membranes. Muscarinic receptor (mAChR) density, as determined by [3H]-QNB binding decreased slightly in cortical membranes, but not in hippocampal membranes. These data suggest that muscarinic stimulated [3H]-PtdIns responses are decreased in senescent brain primarily due to an uncoupling of the receptor-G-protein and/or G-protein-PLC link, although decreases in receptor density may also contribute to reduced muscarinic [3H]-PtdIns signaling.

Effects of NMDA and ferrous sulfate on oxidation and cell death in primary neuronal cultures.

Excessive oxidative radical production has been implicated in a variety of neurodegerative processes including NMDA (N-methyl-D-aspartate) mediated excitotoxicity. To determine the relationship of oxidation to NMDA-receptor mediated neuronal death, we exposed rat primary cortical neuronal cultures to ferrous sulfate and the fluorescent dyes dichlorofluorescin diacetate (H(2)DCF) and propidium iodide (PI) to monitor reactive oxygen species (ROS) and cell death, respectively in the same cultures. Ferrous sulfate (FeSO(4)) caused a dose-dependent increase in cellular oxidation with an ED(50) of approximately 136 microM. Levels of oxidation increased over time reaching maximum levels between 15 and 25 min. Ferrous sulfate (ED(50) approximately 241 microM) treatment for 25 min caused a delayed and progressive neuronal death that was comparable to NMDA (100 microM, 25 min) delayed neuronal death. NMDA (100 microM, 25 min) alone did not result in measurable increases of DCF fluorescence. However, when combined with 40 microM FeSO(4), NMDA dose-dependently increased H(2)DCF fluorescence. Despite the increase in DCF oxidation, combinations of FeSO(4) with NMDA did not synergize or accelerate NMDA-receptor mediated or glutamate-mediated excitotoxicity. Although excessive amounts FeSO(4) induced oxidation can cause delayed neuronal death, these findings suggest that oxidative stress is not the key factor in triggering the NMDA mediated excitotoxic cascade.

Further selection of rat lines differing in 5-HT-1A receptor sensitivity: behavioral and functional correlates.

It was previously reported that selection for differences in the hypothermic effects to the selective 5-HT-1A agonist, 8-OH-DPAT, occurred rapidly, with very substantial differences present by the fourth generation. The present communication summarizes the findings from the next five generations of selection and from behavioral and other functional studies on these rats. The rats which were more sensitive to 8-OH-DPAT (High DPAT Sensitive-HDS) exhibited decreases in temperature of 4 degrees C or more and the distribution did not overlap with that of the rats which were less sensitive to 8-OH-DPAT (Low DPAT Sensitive-LDS) which exhibited decreases in temperature of 1.5 degrees C or less. The randomly bred control group (Random DPAT Sensitive-RDS) exhibited intermediate temperature decreases (means of 1.6-1.8 degrees C), with time overlap with the distributions of the selected groups. Pretreatment with pindolol, a 5-HT-1A antagonist, reduced the hypothermic response to 8-OH-DPAT, but pretreatment with ritanserin, a 5-HT-7 and 5-HT-2A/C antagonist, had no effect, confirming that the hypothermic response to 8-OH-DPAT is mediated predominantly by 5-HT-1A receptors. The HDS rats were less mobile in a forced swim test and drank more saccharin solution in a two-bottle choice paradigm than the LDS or RDS rats over several generations. In contrast, there were no consistent differences among the groups for open field activity or performance in an elevated plus maze. There were no differences among the groups for voluntary alcohol intake, but the HDS rats exhibited greater suppression of alcohol and saccharin intake after injection of 8-OH-DPAT (0.125 mg kg-1). The HDS rats were also found to have a higher number of 5-HT-1A binding sites in cortical regions than the LDS or RDS rats, but there were no 5-HT-1A binding site differences in the raphe nuclei. These findings clearly show that consistent behavioral differences do occur in the 8-OH-DPAT-selected lines of rats, but only for behaviors related to possible depression or reward, not anxiety. The pattern of binding results suggests that these behavioral correlates of 8-OH-DPAT selection may be related to changes in cortical 5-HT-1A receptors rather than raphe autoreceptors.

Concentrations of carbachol stimulating phosphoinositide hydrolysis cause a sustained decrease in membrane potential and firing rate: role of inositol and inositol polyphosphate second messengers.

We have investigated the relationship between muscarinic agonist-stimulated phosphoinositide (PI) hydrolysis and electrophysiological responses in rat hippocampal slice preparations. In a previous extracellular study, we found that muscarinic agonists at concentrations that stimulate PI hydrolysis result in a biphasic firing response; an initial increase in firing followed by loss of firing at higher concentrations. To test the hypothesis that variability in obtaining consistent loss of firing is related to depletion of intracellular inositol, we investigated the effects of adding exogenous inositol to the buffer. We now report that concentrations of inositol similar to those in cerebral spinal fluid (30-100 microM) augment carbamylcholine (carbachol, CCh) mediated loss of firing and [3H]inositol-1,3,4,5-tetrakisphosphate ([3H]Ins(1,3,4,5)P4) formation. Inhibition of firing produced by 30 microM CCh in the presence of inositol was associated with a sustained depolarization of 20-25 mV, an increased slope resistance in the depolarized range (-60 to -40 mV), and a parallel shift in the hyperpolarized (-100 to -70 mV) range of the voltage-current curve and increased frequency of spontaneous IPSPs. Under voltage-clamp, measurements of the M-current (IM) showed sustained inactivation by CCh with reversal after washout of CCh. Manual depolarization of cells by current injection to the same level of depolarization as attained with CCh did not usually lead to the same loss of firing. These findings suggest that IM, and possibly other voltage-independent currents or ion pumps, may cause loss of firing only in part through a depolarization blockade of firing and not through desensitization. Furthermore, CCh treatment without inositol did not depolarize neurons as much as CCh with inositol, and usually did not cause a delayed loss of firing. Brain slice preparations may thus require physiological concentrations of inositol to show consistent or maximum phosphoinositide-mediated electrophysiological responses.