Samuel G. Madamba

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.

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.

Reduced long-term potentiation in the dentate gyrus of transgenic mice with cerebral overexpression of interleukin-6☆

The cytokine interleukin-6 (IL-6) may be a contributing mediator of CNS injury in several neurological disorders. To investigate the role of IL-6 in memory-related disorders, we examined transgenic mice (GFAP-IL6) with cerebral overexpression of IL-6 using paired-pulse facilitation, paired-pulse inhibition, and long-term potentiation (LTP) in an in vitro preparation. We found that paired-pulse potentiation and inhibition in the dentate gyrus of hippocampal slices prepared from the GFAP-IL6 mice did not differ significantly from age-matched control animals, suggesting that the increase in paired-pulse inhibition seen previously in in vivo studies of this model was due to alterations of afferents from other brain regions. However, LTP in the dentate was significantly reduced in slices from GFAP-IL6 transgenic mice when compared with littermate wild-type controls, providing support for a role of IL-6 in the pathogenesis of neurodegenerative associated memory-related disorders.

Chronic ethanol exposure and protracted abstinence alter NMDA receptors in central amygdala

We recently reported that chronic ethanol treatment (CET) and early withdrawal (2–8 h) altered glutamatergic transmission at both pre- and postsynaptic sites in central nucleus of the amygdala (CeA). Acute ethanol (44 mM) inhibited the NMDA receptor (NMDAR)-mediated EPSCs (NMDA-EPSCs) more in CeA neurons from CET rats than from naïve rats and also decreased paired-pulse facilitation (PPF) of NMDA-EPSCs only in CET rats. To determine whether these CET effects persisted after prolonged withdrawal, we recorded intracellularly in rat CeA slices and measured mRNA and protein expression of CeA NMDAR subunits from CET rats and those withdrawn from ethanol for 1 or 2 weeks. At 1 week withdrawal, acute ethanol decreased evoked NMDA-EPSC amplitudes and NMDA currents induced by exogenous NMDA (∼20%) equally to that in naïve rats, indicating that CET effects on postsynaptic mechanisms reversed 1 week after CET cessation. However, acute ethanol still decreased PPF of NMDA-EPSCs, indicating that the acute ethanol-induced increase in glutamate release in CeA seen in CET rats was still present at this time. CET also significantly increased mRNA levels of NR1 and NR2B NMDAR subunits compared to control rats. At 1 week withdrawal, mRNA levels for NR1 and NR2B subunits were significantly decreased. These changes reversed at 2 weeks withdrawal. In Western blots, a significant increase in protein for all three subunits occurred in CeA from CET rats, but not after 1 and 2 weeks of withdrawal. These data indicate that CET induces reversible neuroadaptations in synaptic function, gene expression, and protein composition of NMDAR at CeA synapses.

Arachidonate 5-lipoxygenase and its activating protein: Prominent hippocampal expression and role in somatostatin signaling

Abstract: 5‐Lipoxygenase‐activating protein (FLAP) is an 18‐kDa integral membrane protein required, in peripheral cells, for the activation of 5‐lipoxygenase (5‐LO) and for the resulting synthesis of leukotrienes from arachidonic acid. In the brain, the leukotrienes have been implicated in several pathophysiological events and in the electrophysiological effect of somatostatin, yet the cellular origin and role of these messenger molecules are still poorly understood. In the present study, we used reverse transcriptase‐polymerase chain reaction, in situ hybridization, and immunohistochemistry to demonstrate that 5‐LO and FLAP are expressed in various regions of the rat brain, including hippocampus, cerebellum, primary olfactory cortex, superficial neocortex, thalamus, hypothalamus, and brainstem. Highest levels of expression were observed in cerebellum and hippocampus. In the latter we demonstrate the colocalization of 5‐LO and FLAP in CA1 pyramidal neurons. Moreover, electrophysiological experiments show that selective inhibition of FLAP with the compound MK‐886 (0.25–1 µM) prevents the somatostatin‐induced augmentation of the hippocampal K+ M‐current. Our results provide necessary evidence for the presence and signaling role of 5‐LO and FLAP in central neurons and strongly support their proposed participation in somatostatin‐receptor transmembrane signaling.

Opioid peptides reduce synaptic transmission in the nucleus accumbens

Behavioral studies implicate the nucleus accumbens (NAcc) as a brain area pivotal for the rewarding effects of opiates like heroine and morphine. Therefore, we studied the effect of a variety of opioids on membrane properties and responses to synaptic stimulation in a slice preparation of the NAcc using intracellular recording. Superfusion of opioid peptides did not affect the membrane potential or input resistance of NAcc neurons, but significantly reduced both depolarizing and hyperpolarizing synaptic potentials. Naloxone superfusion significantly reversed the depressant effects of the mu and delta receptor agonists (but not those of the kappa agonist) on synaptic transmission, suggesting involvement of opiate receptors. These results imply that the predominant effect of opiates in NAcc is a reduction of synaptic transmission.

Chronic morphine treatment alters expression of N‐methyl‐D‐aspartate receptor subunits in the extended amygdala

The nucleus accumbens (NAcc) and central amygdala (CeA) are parts of the extended amygdala, a complex that plays a key role in drug abuse and dependence. Our previous studies showed that opiates and ethanol alter glutamatergic transmission in these regions. N‐methyl‐D‐aspartate (NMDA) receptors are key components of glutamatergic transmission likely involved in the development of opiate tolerance and dependence. In this study we examined the effects of chronic morphine administration on gene and protein expression of three major NMDA receptors subunits (NR1, NR2A, and NR2B) in NAcc and CeA. Real‐time PCR showed no differences in mRNA levels of any of the subunits in the whole NAcc between naïve and morphine‐dependent rats. However, at the protein level, immunoblotting revealed that chronic morphine significantly increased levels of NR1 and NR2B subunits. In contrast to the case for NAcc, in CeA we found an increased mRNA level for the NR1 subunit only but unchanged protein levels of all three subunits in morphine‐dependent rats. The altered expressions of NMDA receptor subunits, especially in NAcc, of morphine‐dependent rats may represent a neuroadaptation to chronic morphine and suggest a mechanism for the changes of glutamatergic transmission found in the extended amygdala in dependent rats. In addition, our results indicate a region‐specific response of NMDA receptor subunits to chronic morphine administration at the gene and protein levels.