Carl L. Faingold

Ethanol and neurotransmitter interactions—from molecular to integrative effects

There is extensive evidence that ethanol interacts with a variety of neurotransmitters. Considerable research indicates that the major actions of ethanol involve enhancement of the effects of gamma-aminobutyric acid (GABA) at GABAA receptors and blockade of the NMDA subtype of excitatory amino acid (EAA) receptor. Ethanol increases GABAA receptor-mediated inhibition, but this does not occur in all brain regions, all cell types in the same region, nor at all GABAA receptor sites on the same neuron, nor across species in the same brain region. The molecular basis for the selectivity of the action of ethanol on GaBAA receptors has been proposed to involve a combination of benzodiazepine subtype, beta 2 subunit, and a splice variant of the gamma 2 subunit, but substantial controversy on this issue currently remains. Chronic ethanol administration results in tolerance, dependence, and an ethanol withdrawal (ETX) syndrome, which are mediated, in part, by desensitization and/or down-regulation of GABAA receptors. This decrease in ethanol action may involve changes in subunit expression in selected brain areas, but these data are complex and somewhat contradictory at present. The sensitivity of NMDA receptors to ethanol block is proposed to involve the NMDAR2B subunit in certain brain regions, but this subunit does not appear to be the sole determinant of this interaction. Tolerance to ethanol results in enhanced EAA neurotransmission and NMDA receptor upregulation, which appears to involve selective increases in NMDAR2B subunit levels and other molecular changes in specific brain loci. During ETX a variety of symptoms are seen, including susceptibility to seizures. In rodents these seizures are readily triggered by sound (audiogenic seizures). The neuronal network required for these seizures is contained primarily in certain brain stem structures. Specific nuclei appear to play a hierarchical role in generating each stereotypical behavioral phases of the convulsion. Thus, the inferior colliculus acts to initiate these seizures, and a decrease in effectiveness of GABA-mediated inhibition in these neurons is a major initiation mechanism. The deep layers of superior colliculus are implicated in generation of the wild running behavior. The pontine reticular formation, substantia nigra and periaqueductal gray are implicated in generation of the tonic-clonic seizure behavior. The mechanisms involved in the recruitment of neurons within each network nucleus into the seizure circuit have been proposed to require activation of a critical mass of neurons. Achievement of critical mass may involve excess EAA-mediated synaptic neurotransmission due, in part, to upregulation as well as other phenomena, including volume (non-synaptic diffusion) neurotransmission. Effects of ETX on receptors observed in vitro may undergo amplification in vivo to allow the excess EAA action to be magnified sufficiently to produce synchronization of neuronal firing, allowing participation of the nucleus in seizure generation. GABA-mediated inhibition, which normally acts to limit excitation, is diminished in effectiveness during ETX, and further intensifies this excitation.

Involvement of GABA in acoustically-evoked inhibition in inferior colliculus neurons.

Most criteria for establishing GABA as an inhibitory neurotransmitter in the central nucleus of inferior colliculus (ICc) have been satisfied, but the role of GABA in acoustic coding in ICc is not established. The present study examined this issue by evaluating the effects of iontophoretic application of agents that alter activity at GABA receptors on potential forms of acoustically-evoked inhibition in ICc neurons. Application of the GABAA antagonist, bicuculline, selectively blocked the firing reduction at high intensities observed during non-monotonic rate-intensity functions in ICc neurons. Binaural inhibition was selectively blocked by bicuculline and increased by nipecotic acid. Application of GABA, nipecotic acid (GABA uptake inhibitor) and a benzodiazepine (flurazepam), which enhances the action of GABA, increased the duration and intensity of ipsilateral inhibition and response pause, while bicuculline blocked these acoustically-evoked inhibitory events. Offset inhibition was increased by nipecotic acid application and reduced by bicuculline with the appearance of an offset peak. The present data support an important role for GABA as a neurotransmitter, mediating, in part, non-monotonicity, binaural inhibition, response pause and offset inhibition in ICc neurons. Alterations of these GABA-mediated inhibitory phenomena may occur in auditory dysfunctions observed with aging and audiogenic seizures.

On the role of GABA as an inhibitory neurotransmitter in inferior colliculus neurons: iontophoretic studies

Significant neurochemical, immunocytochemical, and ligand binding studies support a role for GABA as an inhibitory neurotransmitter in the inferior colliculus (IC). The present study attempted to satisfy some of the remaining criteria for establishing transmitter identity by utilizing iontophoretic application onto IC neurons of agents affecting the action of gamma-aminobutyric acid (GABA). The agents examined include GABA, a GABAB agonist (baclofen), a GABAA antagonist (bicuculline), a GABA uptake inhibitor (nipecotic acid), and a benzodiazepine (flurazepam), thought to exert its actions on the GABA receptor complex. Application of GABA results in inhibition of the spontaneous firing and acoustically evoked responses of inferior colliculus neurons. The inhibitory effect of GABA is enhanced by the simultaneous application of nipecotic acid or flurazepam. These agents as well as baclofen produce firing reductions when applied alone in higher doses. The effect of GABA can be blocked by application of bicuculline, and acoustically evoked (binaural) inhibition can also be selectively blocked by low doses of this GABAA antagonist. These data along with previous studies utilizing different techniques fulfill many of the criteria for establishment of GABA as an important inhibitory transmitter in the inferior colliculus.

Induction of audiogenic seizure susceptibility by focal infusion of excitant amino acid or bicuculline into the inferior colliculus of normal rats.

N-Methyl-D-aspartate (NMDA) (10 to 20 nmol) or bicuculline (15 to 50 pmol) in 0.5 microliter was infused bilaterally into the inferior colliculus or the deep layers of superior colliculus (DLSC) in normal rats, and the response to high intensity acoustic stimulation was examined. Thirty-five percent of rats receiving NMDA infusions and 42% of animals receiving bicuculline infusions into the inferior colliculus exhibited sound-induced seizures exclusively that were behaviorally similar to audiogenic seizures displayed by genetically epilepsy-prone rats. Rats receiving microinjections into the DLSC did not display sound-specific seizures. A combined pattern of spontaneous and sound-induced seizures was seen in some rats with both drugs and loci of microinjection. These data and previous studies support a role for increased excitant amino acid action and decreased efficacy of GABA in the inferior colliculus as important mechanisms involved in genetic susceptibility to audiogenic seizures.

The genetically epilepsy-prone rat

1. The genetically epilepsy-prone rat (GEPR) is a valuable model for investigating mechanisms involved in epilepsy because of the controllable nature of the convulsions and their genetic origin. 2. The GEPR exhibits audiogenic seizures (AGS) and also displays higher than normal sensitivity to convulsant drugs, kindling, electroshock and hyperthermic seizures. 3. An abnormal electroencephalographic pattern and increased thresholds for auditory evoked potentials from the cochlea and brainstem are observed in the GEPR. 4. Afterdischarge-like responses and decreased sound-induced inhibition are observed in neurophysiological recordings from neurons of the inferior colliculus (IC) in the GEPR. 5. Significant deficits of norepinephrine and serotonin are observed in many regions of the GEPR brain. 6. Increases in the number of GABAergic neurons and a reduced effectiveness of iontophoretically-applied GABA are observed in the IC of this animal. 7. GABA agonists or an excitant amino acid (EAA) antagonist block AGS susceptibility when microinjected into brainstem auditory nuclei of the GEPR up to the level of IC. 8. A GABA antagonist or an EAA agonist induces susceptibility to AGS in normal rats following microinjection into IC. An increase in EAA release in IC during AGS in the GEPR is also observed. 9. This increased release of EAA and the reduced effectiveness of GABA in IC may be important seizure initiation mechanisms in the GEPR. 10. The AGS pathway in the GEPR appears to involve the auditory nuclei up to the IC as well as the brainstem reticular formation and substantia nigra but not the entopenduncular nucleus or hippocampus.

Decreased effectiveness of GABA-mediated inhibition in the inferior colliculus of the genetically epilepsy-prone rat.

The inferior colliculus (IC) is a critical site for induction of audiogenic seizures in the genetically epilepsy-prone rat (GEPR). Abnormal response properties observed in inferior colliculus neurons of that strain include a high incidence of onset-offset responses which may be a form of afterdischarge. These response abnormalities may involve altered actions of neurotransmitters in that region of the brain. GABA is implicated as a transmitter in endogenous sound-induced inhibition in the inferior colliculus. Endogenous inhibition and the actions of agents that affect GABA receptors were examined in inferior colliculus neurons in epileptic and normal rats. The iontophoretic dose (current) of GABA required to suppress neuronal firing in the epilepsy-prone rat was significantly greater than that required in neurons of the normal rat. A form of endogenous (binaural) inhibition in inferior colliculus neurons, which is proposed to be GABA-mediated, was also significantly reduced in the epilepsy-prone rat as compared with the normal rat. A benzodiazepine (flurazepam) which enhances the action of GABA in many brain sites including the inferior colliculus was significantly less effective than normal when applied iontophoretically onto the same neurons of the epilepsy-prone rat. The GABAA antagonist, bicuculline, which blocks the effect of GABA on inferior colliculus neurons, frequently induced the onset-offset response in neurons not previously exhibiting this pattern. These findings suggest that the reduction of GABA-mediated inhibition in the genetically epilepsy-prone rat may result in the increased incidence of afterdischarges in neurons of the inferior colliculus and may serve as an important mechanism of epileptogenesis in audiogenic seizures in this genetic form of epilepsy.

Repetition of audiogenic seizures in genetically epilepsy-prone rats induces cortical epileptiform activity and additional seizure behaviors

Repetition of seizures appears to increase severity in a number of seizure models, but the nature of these severity increases has not been elucidated in naturally occurring genetic epilepsy models. The genetically epilepsy-prone rat (GEPR) is highly susceptible to many seizure provoking stimuli, and high intensity acoustic stimuli induce audiogenic seizures (AGS). The role of forebrain structures in AGS in the GEPR has not been clear, and the presence of cortical epileptiform EEG activity in the GEPR is controversial. The present study examined the effects of 21 daily AGS repetitions on behavior and EEG activity recorded from the cortex of two GEPR substrains that exhibit moderate (GEPR-3) or severe AGS (GEPR-9). The results indicated that AGS repetition induced seizure severity increases in both GEPR substrains and resulted in prominent cortical epileptiform EEG activity. The AGS behavioral patterns remained distinctly different in the two substrains throughout seizure repetition. In each substrain a different additional behavioral phase was expressed; the GEPR-9 exhibited post-tonic clonus, and the GEPR-3 exhibited facial and forelimb clonus. These findings indicate that seizure repetition results in expansion of the neuronal network subserving AGS to involve forebrain structures. The medial geniculate body and amygdala appear to be part of this expanded network, and long-term potentiation, which was reported in the pathway between the latter brain structures, may be involved. These data suggest that recruitment of forebrain structures into the AGS neuronal network appears to be essential for production of the additional ictal behaviors evoked by AGS repetition.

Evidence supporting a role of serotonin in modulation of sudden death induced by seizures in DBA/2 mice.

Summary:  Purpose: Sudden unexpected death in epilepsy (SUDEP) is a serious concern for epilepsy patients. DBA/2 mice are proposed as a SUDEP model, because these mice exhibit respiratory arrest (RA) after audiogenic seizures (AGSs), and RA is also implicated in human SUDEP. Respiratory mechanisms are modulated, in part, by serotonin. Therefore we evaluated the effects of serotoninergic agents on RA incidence in DBA/2 mice.

Excitant amino acids and audiogenic seizures in the genetically epilepsy-prone rat. II. Efferent seizure propagating pathway ☆

The afferent pathway involved in initiation of audiogenic seizures in the genetically epilepsy-prone rat was investigated by bilateral microinfusion of the excitant amino acid antagonist 2-amino-7-phosphonoheptanoate into the major brain stem and subcortical nuclei of the auditory system. This antagonist has been shown to possess anticonvulsant properties in other seizure models, and an excitant amino acid has been implicated as a putative neurotransmitter in several of these nuclei. Seizure severity was significantly reduced following infusion of this agent into the cochlear nucleus, superior olivary complex, inferior colliculus, and medial geniculate body. Many of these animals exhibited a complete blockade of seizures. The smallest effective dose in the cochlear nucleus and the medial geniculate body was 5 nmol per side. The smallest effective dose in the olive was 1 nmol, and in the inferior colliculus 0.1 nmol per side was protective. The onset of anticonvulsant effectiveness was earliest in the inferior colliculus. These findings showed that the inferior colliculus was the most sensitive auditory center to the anticonvulsant action of 2-amino-7-phosphonoheptanoate and that imbalance between inhibitory and excitatory transmission within this brain structure may be crucial in the initiation of audiogenic seizures in the genetically epilepsy-prone rat.

Stimulation or blockade of the dorsal nucleus of the lateral lemniscus alters binaural and tonic inhibition in contralateral inferior colliculus neurons

Recent studies have demonstrated that several specific types of acoustically-evoked GABA-mediated inhibition occur in neurons of the central nucleus of inferior colliculus (ICc). The dorsal nucleus of the lateral lemniscus (DNLL) provides a major GABAergic projection to ICc. The present study examined the effects of electrical or chemical stimulation or reversible blockade within the DNLL on the discharge characteristics of ICc neurons in anesthetized rats. Microinjection of a local anesthetic (lidocaine) or a GABA-A agonist (THIP) via a cannula placed into DNLL reversibly blocked acoustically-evoked binaural inhibition and increased spontaneous firing in most contralateral ICc neurons. Trains of electrical pulses or microinjection of the excitant amino acid, kainate, into DNLL resulted in reduced acoustically-evoked firing, which was similar to binaural inhibition, in most contralateral ICc neurons examined. The effects of DNLL electrical stimulation were reversibly blocked by microinjection of THIP into the stimulation site, suggesting that the effect of the electrical stimulation is mediated by direct effects on cell bodies of DNLL neurons. These data support the idea that contralateral GABAergic input from the DNLL is inhibitory to ICc neurons. Thus, binaural inhibition and tonic inhibition in ICc neurons may be mediated, in part, by the GABAergic projection from the contralateral DNLL.