George R. Siggins

Studies on norepinephrine-containing afferents to Purkinje cells of rat cerebellum. II. Sensitivity of Purkinje cells to norepinephrine and related substances administered by microiontophoresis.

Abstract The sensitivity of rat cerebellar Purkinje cells to norepinephrine and to adrenergic antagonists and protagonists was studied by microiontophoresis. (1) Virtually all Purkinje cells respond to microiontophoresis of norepinephrine with a reduction in mean spontaneous discharge rate. (2) The effects of norepinephrine are reproduced by epinephrine, isoproterenol, amphetamine, and tyramine. (3) Responses to norepinephrine are blocked by the beta receptor antagonist, MJ-1999. (4) Desmethylimipramine, which blocks re-uptake of norepinephrine, markedly potentiates the response to this agent. (5) Norepinephrine, tyramine, amphetamine and MJ-1999 are effective in animals treated with 6-hydroxydopamine, indicating that their effects are not exerted on adrenergic presynaptic terminals. (6) These data support the hypothesis that norepinephrine is a Purkinje cell neurotransmitter.

The ontogenetic development of synaptic junctions, synaptic activation and responsiveness to neurotransmitter substances in rat cerebellar purkinje cells

Ultrastructural quantitation of synaptogenesis in cerebellum showed the onset of contacts stained by ethanolic phosphotungstic acid at days 3 and 4 with a progressive increase in density of such contacts at later ages. Electrophysiological studies revealed that both responses to parallel fiber stimulation and the occurence of climbing fiber burst responses appear late in the third postnatal day. Studies of drug sensitivity employing microiontophoretic techniques showed that spontaneous activity of Purkinje cells between 0 and 4 postnatal days is accelerated by glutamate, and inhibited by gamma-aminobutyric acid, norepinephrine, adenosine cyclic monophosphate (cyclic AMP) and dinitrophenol. Prostaglandin E1 antagonized the action of norepinephrine. These studies indicate that the membrane chemosensitivity to the adult spectrum of putative transmitters precedes the onset of synaptogenesis.

Studies on norepinephrine-containing afferents to Purkinje cells of rat cerebellum. I. Localization of the fibers and their synapses

A process is described for the purification and recovery of cyanuric acid from a hot slurry of cyanuric acid in an organic solvent. In the process, the hot slurry is quenched in a quench liquid which is at a temperature for cooling the hot slurry to form a cooled slurry of cyanuric acid particles in a mixture of the solvent and the quench liquid. Cyanuric acid particles are separated from the mixture of solvent and quench liquid and recovered. The process effectively cools and separates cyanuric acid from the solvent and any color bodies present to produce a pure white crystalline product.

Interleukin 1β inhibits synaptic strength and long-term potentiation in the rat CA1 hippocampus

Cytokines such as interleukin-1 beta (IL-1 beta) are released in the nervous system following inflammation or infection. Recently, IL-1 beta was shown to enhance synaptic inhibitory mechanisms. We therefore investigated the effect of IL-1 beta superfusion on long-term potentiation (LTP), the cellular model of memory and learning, evoked in the CA1 region by tetanic stimulation of the stratum radiatum in the rat hippocampal slice. IL-1 beta (150 pM-1.5 nM) superfused 10 min before tetanic stimulation significantly reduced LTP of the slope of the population excitatory postsynaptic potential (pEPSP) and the population spike (PS) amplitude in CA1 in a concentration-dependent manner. IL-1 beta (1.5 nM) applied for 10 min 1 h before tetanus significantly inhibited LTP of the PS amplitude and pEPSP slope and reduced pEPSP and PS values before tetanus as well, although the PS returned to control values before tetanus. Heat-inactivated IL-1 beta had no effect on pre-tetanus pEPSP or PS values or the induction of LTP. These data demonstrate that IL-1 beta modulates synaptic potentials and reduces LTP. These findings have important implications for the role of IL-1 beta in neuronal disorders following infection, perhaps best exemplified by HIV-1-associated dementia.

Ethanol increases GABAergic transmission at both pre- and postsynaptic sites in rat central amygdala neurons

We examined the interaction of ethanol with the γ-aminobutyric acid (GABA)ergic system in neurons of slices of the rat central amygdala nucleus (CeA), a brain region thought to be critical for the reinforcing effects of ethanol. Brief superfusion of 11–66 mM ethanol significantly increased GABA type A (GABAA) receptor-mediated inhibitory postsynaptic potentials (IPSPs) and currents (IPSCs) in most CeA neurons, with a low apparent EC50 of 20 mM. Acute superfusion of 44 mM ethanol increased the amplitude of evoked GABAA IPSPs and IPSCs in 70% of CeA neurons. The ethanol enhancement of IPSPs and IPSCs occurred to a similar extent in the presence of the GABA type B (GABAB) receptor antagonist CGP 55845A, suggesting that this receptor is not involved in the ethanol effect on CeA neurons. Ethanol superfusion also decreased paired-pulse facilitation of evoked GABAA IPSPs and IPSCs and always increased the frequency and sometimes the amplitude of spontaneous miniature GABAA IPSCs as well as responses to local GABA application, indicating both presynaptic and postsynaptic sites of action for ethanol. Thus, the CeA is the first brain region to reveal, without conditional treatments such as GABAB antagonists, consistent, low-dose ethanol enhancement of GABAergic transmission at both pre- and postsynaptic sites. These findings add further support to the contention that the ethanol–GABA interaction in CeA plays an important role in the reinforcing effects of ethanol.

Acute and Chronic Ethanol Alter Glutamatergic Transmission in Rat Central Amygdala: an In Vitro and In Vivo Analysis

The modulation of glutamatergic transmission by ethanol may contribute to ethanol intoxication, reinforcement, tolerance, and dependence. Therefore, we used in vitro electrophysiological and in vivo microdialysis techniques to investigate the effects of acute and chronic ethanol on glutamatergic transmission in the central nucleus of amygdala (CeA). Superfusion of 5-66 mm ethanol decreased compound glutamatergic EPSPs and EPSCs in CeA neurons, with half-maximal inhibition elicited by 14 mm ethanol. Ethanol (44 mm) decreased both non-NMDAR- and NMDAR-mediated EPSPs and EPSCs by 21%. Both the ethanol- and ifenprodil-induced depression of NMDAR-mediated EPSPs and EPSCs was enhanced in rats that received chronic ethanol treatment (CET). Ifenprodil also occluded the ethanol effect, suggesting that NR2B subunit-containing receptors may be involved. With local applications of NMDA, acute ethanol elicited a greater inhibition of NMDA currents in slices taken from CET (47%) compared with naive (30%) animals, suggesting that CET sensitizes NMDA receptors to ethanol. Acute ethanol also reduced paired pulse facilitation of EPSPs and EPSCs only in CET animals, suggesting acute ethanol-induced increase of glutamate release. This finding was supported by in vivo experiments showing that infusion of ethanol (0.1-1 m) via reverse microdialysis significantly increased glutamate release into the CeA dialysate but only after CET. Moreover, baseline CeA glutamate content was significantly higher in CET compared with naive animals. These combined findings suggest that CET and withdrawal lead to neuroadaptations of glutamatergic transmission at both presynaptic and postsynaptic sites in CeA, and glutamatergic synapses in CeA may play an important role in ethanol dependence.

Studies on norepinephrine-containing afferents to Purkinje cells of rat cerebellum. III. Evidence for mediation of norepinephrine effects by cyclic 3′, 5′-adenosine monophosphate

Abstract The relationship between cerebellar adenyl cyclase and norepinephrine (NE) receptivity of cerebellar Purkinje cells was explored by recording their spontaneous discharge during microiontophoretic application of various substances. The depression in firing rate of single Purkinje cells produced by NE was usually mimicked by adenosine 3′5′-monophosphate (cyclic AMP). The synthetic dibutyryl derivative of cyclic AMP, however, was no more potent than the parent compound. Two other adenine nucleotides, adenosine triphosphate (ATP) and 5′-AMP generally accelerated discharge rate or had no effect. The excitatory effects of the nucleotides may be related to chelation of divalent cations, since EDTA and citrate were universally found to accelerate unit firing while calcium ions always slowed or blocked spontaneous activity. Further evidence for a link between the effects of NE and cyclic AMP was seen with the administration of the methyl xanthines, which inhibit the enzymatic breakdown of cyclic AMP by phosphodiesterase. Theophylline and aminophylline, whether parenterally or iontophoretically administered, markedly potentiated the depressant effects of both NE and cyclic AMP on Purkinje cells. Methyl xanthines usually transformed the infrequent speeding effects of cyclic AMP to those of slowing, or brought about an inhibitory response in cells previously non-responsive to cyclic AMP. Furthermore, iontophoresis of prostaglandins E1 and E2 (but not F1α, F2α, linoleic or linolenic acids), and of nocotinate, reported to reduce cyclic AMP levels in peripheral neuro-effector systems, also block the action of iontophoretically applied NE in rat cerebellum. To determine if the locus of the action of the various iontophoretically applied substances was exerted presynaptically on the proposed adrenergic terminals on Purkinje cells, 6-hydroxydopamine (6-HODA) treated animals were studied. Under these conditions the effects of all the drugs and their interactions appeared identical to those of normal cerebella, in spite of biochemical and histochemical evidence for complete degeneration of adrenergic nerve terminals. These studies provide indirect evidence for mediation of NE effects by cyclic AMP in a living single cell system. Thus, they parallel in vitro studies with cerebellar slices which show an increase in cyclic AMP levels with NE, as well as histamine. Taken with the histochemical data for norepinephrine nerves terminating on Purkinje cells, the findings point to a possible transsynaptic modulation of adenyl cyclase activity.

Cyclic Adenosine Monophosphate and Norepinephrine: Effects on Transmembrane Properties of Cerebellar Purkinje Cells

Electrical properties of Purkinje cells were recorded by intracellular microelectrode during extracellular electrophoretic application of gamma aminobutyrate, norepinephrine, cyclic adenosine monophosphate, and dibutyryl cyclic adenosine monophosphate. All these substances hyperpolarized Purkinje cells. Transmembrane resistance decreased during gamma aminobutyrate hyperpolarization. In contrast, norepinephrine and the cyclic nucleotides generally elevated resistance. These results show that cyclic nucleotides mimic the unique effects of norepinephrine on the bioelectric properties of neuronal membranes.

Corticotropin Releasing Factor–Induced Amygdala Gamma-Aminobutyric Acid Release Plays a Key Role in Alcohol Dependence

BACKGROUND Corticotropin-releasing factor (CRF) and gamma-aminobutyric acid (GABA)ergic systems in the central amygdala (CeA) are implicated in the high-anxiety, high-drinking profile associated with ethanol dependence. Ethanol augments CeA GABA release in ethanol-naive rats and mice. METHODS Using naive and ethanol-dependent rats, we compared electrophysiologic effects and interactions of CRF and ethanol on CeA GABAergic transmission, and we measured GABA dialyzate in CeA after injection of CRF(1) antagonists and ethanol. We also compared mRNA expression in CeA for CRF and CRF(1) using real-time polymerase chain reaction. We assessed effects of chronic treatment with a CRF(1) antagonist on withdrawal-induced increases in alcohol consumption in dependent rats. RESULTS CRF and ethanol augmented CeA GABAergic transmission in naive rats via increased GABA release. Three CRF1 receptor (CRF(1)) antagonists decreased basal GABAergic responses and abolished ethanol effects. Ethanol-dependent rats exhibited heightened sensitivity to CRF and CRF(1) antagonists on CeA GABA release. Intra-CeA CRF(1) antagonist administration reversed dependence-related elevations in GABA dialysate and blocked ethanol-induced increases in GABA dialyzate in both dependent and naive rats. Polymerase chain reaction studies indicate increased expression of CRF and CRF(1) in CeA of dependent rats. Chronic CRF(1) antagonist treatment blocked withdrawal-induced increases in alcohol drinking by dependent rats and tempered moderate increases in alcohol consumption by nondependent rats in intermittent testing. CONCLUSIONS These combined findings suggest a key role for specific presynaptic CRF-GABA interactions in CeA in the development and maintenance of ethanol dependence.

Increased GABA Release in the Central Amygdala of Ethanol-Dependent Rats

The central nucleus of amygdala (CeA) is important in regulating alcohol consumption and plays a major role in the anxiogenic response to ethanol withdrawal. We showed previously that acute ethanol augments GABAA receptor-mediated IPSPs and IPSCs, possibly by a presynaptic mechanism. Here, we have examined the interaction of acute ethanol with the GABAergic system in chronic ethanol-treated (CET) rats using an in vitro CeA slice preparation and in vivo brain microdialysis. We found that in CeA slices from CET rats, the baseline evoked IPSP and IPSC amplitudes were increased, and paired-pulse facilitation ratios were lower than in naive rats, suggesting an increased GABAergic transmission after chronic ethanol treatment. Interestingly, acute ethanol (5-66 mm) significantly enhanced IPSPs and IPSCs equally in CET and naive rats, indicating a lack of tolerance for this effect of acute ethanol. Analysis of miniature IPSC frequency suggests that the increased GABAergic transmission by both acute and chronic ethanol arises from a presynaptic mechanism involving enhanced vesicular release of GABA. These data are supported by microdialysis studies showing that CET rats presented a fourfold increase in baseline GABA dialysate content compared with naive rats. In vivo administration of ethanol (0.1, 0.3, and 1.0 m) produced a dose-dependent increase in GABA release in the CeA dialysate in both CET and naive rats. These combined findings suggest that acute and chronic ethanol increases GABA release in CeA and support previous reports that the behavioral actions of ethanol are mediated, in part, by increased GABAergic transmission in the CeA.