Reticular nucleus-specific changes in α3 subunit protein at GABA synapses in genetically epilepsy-prone rats

Abstract

Differential composition of GABAA receptor (GABAAR) subunits underlies the variability of fast inhibitory synaptic transmission; alteration of specific GABAAR subunits in localized brain regions may contribute to abnormal brain states such as absence epilepsy. We combined immunocytochemistry and high-resolution ImmunoGold electron microscopy to study cellular and subcellular localization of GABA AR ␣1, ␣3, and ␤2/␤3 subunits in ventral posterior nucleus (VP) and reticular nucleus (RTN) of control rats and WAG/Rij rats, a genetic model of absence epilepsy. In control rats, ␣1 subunits were prominent at inhibitory synapses in VP and much less prominent in RTN; in contrast, the ␣3 subunit was highly evident at inhibitory synapses in RTN. ␤2/␤3 subunits were evenly distributed at inhibitory synapses in both VP and RTN. ImmunoGold particles representing all subunits were concentrated at postsynaptic densities with no extrasynaptic localization. Calculated mean number of particles for ␣1 subunit per postsynaptic density in nonepileptic VP was 6.1 ؎ 3.7, for ␣3 subunit in RTN it was 6.6 ؎ 3.4, and for ␤2/␤3 subunits in VP and RTN the mean numbers were 3.7 ؎ 1.3 and 3.5 ؎ 1.2, respectively. In WAG/Rij rats, there was a specific loss of ␣3 subunit immunoreactivity at inhibitory synapses in RTN, without reduction in ␣3 subunit mRNA or significant change in immunostaining for other markers of RTN cell identity such as GABA or parvalbumin. ␣3 immunostaining in cortex was unchanged. Subtle, localized changes in GABAAR expression acting at highly specific points in the interconnected thalamocortical network lie at the heart of idiopathic generalized epilepsy.absence epilepsy ͉ inhibition ͉ quantitative electron microscopy T he idiopathic generalized epilepsies are characterized by abrupt losses of consciousness during which the electroencephalogram (EEG) exhibits paroxysmal, high-amplitude spike and wave complexes at Ϸ3Hz and lasting from a few seconds to Ͻ1 min. The loss of consciousness is referred to as an absence seizure or petit mal. Spike and wave activity reflects paroxysmal discharging of neurons in the network of reentrant thalamocortical and corticothalamic connections (1-3). Linkage studies in humans suggest involvement of genes encoding GABA A receptor (GABA A R) subunits of the ␣, ␤, ␥, and ␦ families in absence epilepsy (4-7), and experimental studies in animals point to molecular genetic abnormalities in GABA A R signaling (8, 9). The forms by which genetic anomalies in GABA A R manifest themselves are multiple and varied. They can involve receptor synthesis and trafficking within neurons, changes in ratios of alternatively spliced mRNAs, and translocations of receptor subunits to membrane sites not normally occupied (10-14).The GABAergic neurons of the thalamic reticular nucleus (RTN) play a key role in synchronizing activity in the thalamocortical network during states of consciousness (15, 16), and GABA A R antagonists applied to its cells can transform 7-to 14-Hz sleep spindle oscillations generated in the ...

Lipopolysaccharide induced increase in seizure activity in two animal models of absence epilepsy WAG/Rij and GAERS rats and Long Evans rats

“…There are also similarities with some differences between the 3 strains as far as the mechanisms of SWDs. It has been demonstrated, that (i) several neurotransmitter systems are involved in the pathophysiological processes leading to absence epileptic seizures and their maintenance/recurrence such as glutamatergic system, GABAergic system, dopaminergic and nucleosiderg system in GAERS rats, WAG/Rij rats and Long Evans rats (Bazyan and Van Luijtelaar, 2013;Depaulis and Van Luijtelaar, 2005;Kovács et al, 2013Kovács et al, , 2014Li et al, 2006;Polack and Charpier, 2006), (ii) the effects of anti-epileptic drugs on SWDs were similar in GAERS rats, WAG/Rij rats and Long Evans rats (e.g., ethosuximide, and valproate decreased the number of SWDs) (Chen et al, 2011;Depaulis and Van Luijtelaar, 2005;Shaw, 2004Shaw, , 2007, (iii) sodium channel (Nav1.1 and Nav1.6) expression was selectively increased in the somatosensory cortex of WAG/Rij rats in relation to age-dependent increase in seizure number and duration (Klein et al, 2004), (iv) selective decrease in thalamic (e.g., reticular thalamic nucleus) and cortical (e.g., somatosensory cortex) GABA (A) receptor subunits such as reduction of ␣3 subunit of GABA (A) receptor in reticular thalamic nucleus of WAG/Rij rats (Liu et al, 2007) may have a role in the pathophysiology of absence epilepsy in Long Evans rats, WAG/Rij rats and GAERS rats (Li et al, 2006;Spreafico et al, 1993), (v) mRNA levels for most GABA (B(1)) subunits were lower in WAG/Rij neocortex than in cortex of control nonepileptic rats, which can contribute to neocortical hyperexcitability and SWD generation (Merlo et al, 2007) whereas GABA (B(1)) subunit mRNA levels were higher and lower in the somatosensory cortex and ventrobasal thalamic nuclei of GAERS rats, respectively, compared with control animals (Princivalle et al, 2003), (vi) decreased alpha-amino-3-hydroxy-5-methyl-4-isoazolepropionic acid receptor (AMPA; AMPA-GluR4) and N-methyl-d-aspartate receptor (NMDA; NMDA-NR1) expression in the cortical focus of WAG/Rij animals (compared with non-epileptic control rats) and increased AMPA-GluR1/2 expression in GAERS rats in the somatosensory cortex may be in relation to hyperexcitability in somatosensory cortex and to SWD initiation (Van de BovenkampJanssen et al, 2006;Kennard et al, 2011), (vii) reduction of group-I metabotropic glutamate receptors (mGlu1 receptors) in ventrobasal thalamic nuclei and increased expression of group-II metabotropic glutamate receptors (mGlu2/3 receptors) in WAG/Rij rat somatosensory cortex and ventrobasal thalamic nuclei may be involved in the ge...…”

The calcium-binding protein parvalbumin modulates the firing 1 properties of the reticular thalamic nucleus bursting neurons

“…It is known that RTN is characterized by a pattern of collaterals establishing axodendritic, somatodendritic, and dendrodendritic synapses within the RTN (Csillik et al 2005;Spreafico et al 1991;Steriade et al 1984;Yingling and Skinner 1976). These collaterals are GABAergic, expressing GABA(␤3) as opposed to the RTN projections onto the thalamocortical (TC) cells that express GABA(␤3) (Liu et al 2007;Sohal and Huguenard 2003). Recent findings have shown that these collateral synapses within RTN are likely to be much weaker than the RTN-TC synapses (Lam et al 2006).…”

Upholding WAG/Rij rats as a model of absence epileptogenesis: Hidden mechanisms and a new theory on seizure development

“…Human absence epilepsy is associated with mutations in the genes encoding the  5 ,  3 , or  2 subunits of the GABA A receptor (Hirose, 2014) and the amount of the  3 subunit in the NRT has been shown to be reduced in WAG/Rij rats as compared to non-epileptic control rats (Gauguier et al, 2004;Liu et al, 2007); instead, GABA A receptors are preserved in the neocortex of WAG/Rij epileptic rats, which are defective in several GABA B receptor isoforms (Liu et al, 2007;Merlo et al, 2007).…”