Ventrobasal Research Articles

A Cellular & Network Level Investigation of Thalamocortical Neuron Oscillations & the Role of the Transcription Factor, Shox2

Thalamocortical neurons (TCNs) densely innervate the cortex and rhythmically burst fire in synchrony with cortical electrical activity that generates thalamus‐driven sleep spindles. Proper development of thalamocortical neuron cortical connectivity and firing activity underlie these sleep spindles—which are critical for memory consolidation—yet a transcriptional regulator that promotes these critical TCN characteristics has not been identified. Previously, we have shown that the transcriptional factor, Shox2, is important for the expression of key ion channels that generate TCN burst firing. Here, we have developed a targeted model using stereotactic injections of Cre virus into Shox2fl/flmice to isolate the role of Shox2 function in a single thalamic nucleus, the ventrobasal (VB) nucleus, upon TCN burst firing, cortical connectivity and organization, sleep spindles, and ultimately memory consolidation. We utilize patch clamp electrophysiology in slices to analyze burst firing of TCNs, cytochrome oxidase and VGluT2 staining of VB cortical targets (cortical barrels), in vivo electrophysiology analysis of sleep spindles, and novel object recognition to assess the effects of Shox2 KO in VB TCNs. Our findings indicate that the area under the burst curve and number of action potentials is significantly reduced, cortical barrel organization is disrupted in a P6 Shox2 KO model, sleep spindle occurrence is significantly reduced, and novel object recognition is impaired. These results show that Shox2 contributes to regulation of TCN cell properties and overall thalamic function.

Does astrocyte gap junction protein expression differ during development in absence epileptic rats?

Intercellular communication via gap junctions (GJs) has a wide variety of complex and essential functions in the CNS. In the present developmental study, we aimed to quantify the number of astrocytic GJs protein connexin 30 (Cx30) of genetic model of absence epilepsy rats from Strasbourg (GAERS) at postnatal P10, P30, and P60 days in the epileptic focal areas involved in the cortico-thalamic circuit. We compared the results with Wistar rats using immunohistochemistry and western blotting. The number of Cx30 immunopositive astrocytes per unit area were quantified for the somatosensory cortex (SSCx), ventrobasal (VB), and lateral geniculate (LGN) thalamic nuclei of the two strains and Cx30 western blot was applied to the tissue samples from the same regions. Both immunohistochemical and western blot results revealed the presence of Cx30 in all regions studied at P10 in both Wistar and GAERS animals. The SSCx, VB, and LGN of Wistar animals showed progressive increase in the number of Cx30 immunopositive labeled astrocytes from P10 to P30 and reached a peak at P30; then a significant decline was observed from P30 to P60 for the SSCx and VB. However, in GAERS Cx30 immunopositive labeled astrocytes showed a progressive increase from P10 to P60 for all brain regions studied. The immunohistochemical data highly corresponded with western blotting results. We conclude that the developmental disproportional expression of Cx30 in the epileptic focal areas in GAERS may be related to the onset of absence seizures or may be related to the neurogenesis of absence epilepsy.

Comparing astrocytic gap junction of genetic absence epileptic rats with control rats: an experimental study.

The synchronization of astrocytes via gap junctions (GJ) is a crucial mechanism in epileptic conditions, contributing to the synchronization of the neuronal networks. Little is known about the endogenous response of GJ in genetic absence epileptic animal models. We evaluated and quantified astrocyte GJ protein connexin (Cx) 30 and 43 in the somatosensory cortex (SSCx), ventrobasal (VB), centromedian (CM), lateral geniculate (LGN) and thalamic reticular (TRN) nuclei of thalamus of genetic absence epilepsy rats from Strasbourg (GAERS), Wistar albino glaxo rats from Rijswijk (WAG/Rij) and control Wistar animals using immunohistochemistry and Western Blot. The Cx30 and Cx43 immunopositive astrocytes per unit area were quantified for each region of the three animal strains. Furthermore, Cx30 and Cx43 Western Blot was applied to the tissue samples from the same regions of the three strain. The number of Cx30 immunopositive astrocytes showed significant increase in both GAERS and WAG/Rij compared to control Wistar in all brain regions studied except LGN of WAG/Rij animals. Furthermore, Cx43 in both GAERS and WAG/Rij showed significant increase in SSCx, VB and TRN. The protein expression was increased in both Cx30 and Cx43 in the two epileptic strains compared to control Wistar animals. The significant increase in the astrocytic GJ proteins Cx30 and Cx43 and the differences in the co-expression of Cx30 and Cx43 in the genetically absence epileptic strains compared to control Wistar animals may suggest that astrocytic Cx's may be involved in the mechanism of absence epilepsy. Increased number of astrocytic Cx's in GAERS and WAG/Rij may represent a compensatory response of the thalamocortical circuitry to the absence seizures or may be related to the production and/or development of absence seizures.

Photodynamic Modification of Native HCN Channels Expressed in Thalamocortical Neurons

The photodynamic process requires three elements: light, oxygen, and photosensitizer, and involves the formation of singlet oxygen, the molecular oxygen in excited electronic states. Previously, we reported that heterologously expressed hyperpolarization-activated cAMP-gated (HCN) channels in excised membrane patches are sensitive to photodynamic modification (PDM). Here we extend this study to native HCN channels expressed in thalamocortical (TC) neurons in the ventrobasal (VB) complex of the thalamus and dopaminergic neurons (DA) of the ventral tegmental area (VTA). To do this, we introduced the photosensitizer FITC-cAMP into TCs or DAs of rodent brain slices via a whole-cell patch-clamp recording pipette. After illumination with blue light pulses, we observed an increase in the voltage-insensitive, instantaneous Iinst component, accompanied by a long-lasting decrease in the hyperpolarization-dependent Ih component. Both Ih and the increased Iinst after PDM could be blocked by the HCN blockers Cs+ and ZD7288. When FITC and cAMP were dissociated and loaded into neurons as two separate chemicals, light application did not result in any long-lasting changes of the HCN currents. In contrast, light pulses applied to HCN2-/- neurons loaded with FITC-cAMP generated a much greater reduction in the Iinst component compared to that of WT neurons. Next, we investigated the impact of the long-lasting increases in Iinst after PDM on the cellular physiology of VB neurons. Consistent with an upregulation of HCN channel function, PDM elicited a depolarization of the resting membrane potential (RMP). Importantly, Trolox-C, an effective quencher for singlet oxygen, could block the PDM-dependent increase in Iinst and depolarization of the RMP. We propose that PDM of native HCN channels under physiological conditions may provide a photodynamic approach to alleviate HCN channelopathy in certain pathological conditions.

Relationships between astrocytes and absence epilepsy in rat: An experimental study

Astrocytes take part in the modulation of neuronal activity through the uptake and release of both GABA and glutamate. In the present study we aimed to quantify the number of astrocytes expressing the astrocyte marker glial fibrillary acidic protein (GFAP) in the somatosensory cortex (SSCx), ventrobasal (VB), centromedial (CM), reticular (TRN) and dorsal lateral geniculate (dLGN) nuclei of thalamus in Genetic Absence Epilepsy Rats from Strasbourg (GAERS), Wistar Albino Glaxo Rats from Rijswijk (WAG/Rij) and control Wistar animals. Further, we aimed to compare the GFAP protein expression levels between the three animal strains.The GFAP-immunohistochemistry was applied to sections from the SSCx, VB, CM, TRN and dLGN and GFAP-positive astrocytes were quantified for the three animal strains. Further, GFAP Western Blot was applied to the tissue samples from the same regions of the three strain. The data obtained from Wistar animals were compared with GAERS and WAG/Rij animals. The number of GFAP-positive astrocytes per unit area in all brain regions studied showed high significance between Wistar-GAERS and Wistar-WAG/Rij except the dLGN. The GAERS had significant higher endogenous GFAP expression in all brain regions studied compared to Wistar and WAG/Rij animals. These findings demonstrate a discrete difference in both GFAP-positive astrocyte populations and GFAP protein expression levels between Wistar and genetically epileptic strains (GAERS and WAG/Rij). Absence seizures are thought to result from a possible imbalance in glutamatergic and GABAergic neurotransmission. Astrocytes regulate the concentration of glutamate and GABA in the extracellular space in the brain, the difference in the astrocyte population and GFAP protein expression in the epileptic strains clearly shows the involvement of astrocytes in the mechanism of absence epilepsy

Experimentally-constrained biophysical models of tonic and burst firing modes in thalamocortical neurons.

Somatosensory thalamocortical (TC) neurons from the ventrobasal (VB) thalamus are central components in the flow of sensory information between the periphery and the cerebral cortex, and participate in the dynamic regulation of thalamocortical states including wakefulness and sleep. This property is reflected at the cellular level by the ability to generate action potentials in two distinct firing modes, called tonic firing and low-threshold bursting. Although the general properties of TC neurons are known, we still lack a detailed characterization of their morphological and electrical properties in the VB thalamus. The aim of this study was to build biophysically-detailed models of VB TC neurons explicitly constrained with experimental data from rats. We recorded the electrical activity of VB neurons (N = 49) and reconstructed morphologies in 3D (N = 50) by applying standardized protocols. After identifying distinct electrical types, we used a multi-objective optimization to fit single neuron electrical models (e-models), which yielded multiple solutions consistent with the experimental data. The models were tested for generalization using electrical stimuli and neuron morphologies not used during fitting. A local sensitivity analysis revealed that the e-models are robust to small parameter changes and that all the parameters were constrained by one or more features. The e-models, when tested in combination with different morphologies, showed that the electrical behavior is substantially preserved when changing dendritic structure and that the e-models were not overfit to a specific morphology. The models and their analysis show that automatic parameter search can be applied to capture complex firing behavior, such as co-existence of tonic firing and low-threshold bursting over a wide range of parameter sets and in combination with different neuron morphologies.

Abolishing cAMP sensitivity in HCN2 pacemaker channels induces generalized seizures.

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are dually gated channels that are operated by voltage and by neurotransmitters via the cAMP system. cAMP-dependent HCN regulation has been proposed to play a key role in regulating circuit behavior in the thalamus. By analyzing a knockin mouse model (HCN2EA), in which binding of cAMP to HCN2 was abolished by 2 amino acid exchanges (R591E, T592A), we found that cAMP gating of HCN2 is essential for regulating the transition between the burst and tonic modes of firing in thalamic dorsal-lateral geniculate (dLGN) and ventrobasal (VB) nuclei. HCN2EA mice display impaired visual learning, generalized seizures of thalamic origin, and altered NREM sleep properties. VB-specific deletion of HCN2, but not of HCN4, also induced these generalized seizures of the absence type, corroborating a key role of HCN2 in this particular nucleus for controlling consciousness. Together, our data define distinct pathological phenotypes resulting from the loss of cAMP-mediated gating of a neuronal HCN channel.

Photodynamic Modification of Native HCN Channels in Thalamocortical Neurons

Previously, we reported photodynamic modification (PDM) of heterologous expressed HCN channels on membrane patches. In the presence of specific (FITC-cAMP) and non-specific (Rose Bengal) photosensitizers, light excitation transforms the function of HCN channels in an oxygen dependent manner. Here we extended the study to native HCN channels expressed in thalamocortical (TC) neurons in the Ventrobasal (VB) complex in the thalamus. We first discovered that blue light excitation significant increases the current amplitude and the rate of activation of the hyperpolarization-activated Ih current, which was a reversible process and did not require exogenous photosensitizers. However, when FITC-cAMP, but not FITC alone or FITC and cAMP, was loaded into the cell through whole-cell recording pipette, light excitation resulted in long-lasting increases in the voltage-insensitive, instantaneous Iinst component and correspondingly decreases in the Ih component. The Ih and Iinst after PDM can be blocked by Cs+ and ZD7288, confirming the specific involvement of HCN channels. Next, we investigated the impacts of PDM of native HCN channels on the resting membrane potential (RMP) and input resistance (Rin) of VB neurons. As expected, after PDM, there was a significant positive shift in RMP. Importantly, both the long-lasting increase in Iinst and the positive shift in RMP after light excitation, but not the short-term and reversible increase in Ih during light excitation, could be blocked by Trolox-C, a widely used quencher for singlet oxygen. In summary, we report the PDM of native HCN channels under more physiological condition which should carry meanings for both basic researches and clinical treatments of diseases due to HCN channelopathy.

Leptin alters somatosensory thalamic networks by decreasing gaba release from reticular thalamic nucleus and action potential frequency at ventrobasal neurons.

Leptin is an adipose-derived hormone that controls appetite and energy expenditure. Leptin receptors are expressed on extra-hypothalamic ventrobasal (VB) and reticular thalamic (RTN) nuclei from embryonic stages. Here, we studied the effects of pressure-puff, local application of leptin on both synaptic transmission and action potential properties of thalamic neurons in thalamocortical slices. We used whole-cell patch-clamp recordings of thalamocortical VB neurons from wild-type (WT) and leptin-deficient obese (ob/ob) mice. We observed differences in VB neurons action potentials and synaptic currents kinetics when comparing WT vs. ob/ob. Leptin reduced GABA release onto VB neurons throughout the activation of a JAK2-dependent pathway, without affecting excitatory glutamate transmission. We observed a rapid and reversible reduction by leptin of the number of action potentials of VB neurons via the activation of large conductance Ca2+-dependent potassium channels. These leptin effects were observed in thalamocortical slices from up to 5-week-old WT but not in leptin-deficient obese mice. Results described here suggest the existence of a leptin-mediated trophic modulation of thalamocortical excitability during postnatal development. These findings could contribute to a better understanding of leptin within the thalamocortical system and sleep deficits in obesity.

Shank3-deficient thalamocortical neurons show HCN channelopathy and alterations in intrinsic electrical properties.

Shank3 increases the HCN channel surface expression in heterologous expression systems. Shank3Δ13-16 deficiency causes significant reduction in HCN2 expression and Ih current amplitude in thalamocortical (TC) neurons. Shank3Δ13-16 - but not Shank3Δ4-9 -deficient TC neurons share changes in basic electrical properties which are comparable to those of HCN2-/- TC neurons. HCN channelopathy may critically mediate events downstream from Shank3 deficiency. SHANK3 is a scaffolding protein that is highly enriched in excitatory synapses. Mutations in the SHANK3 gene have been linked to neuropsychiatric disorders especially the autism spectrum disorders. SHANK3 deficiency is known to cause impairments in synaptic transmission, but its effects on basic neuronal electrical properties that are more localized to the soma and proximal dendrites remain unclear. Here we confirmed that in heterologous expression systems two different mouse Shank3 isoforms, Shank3A and Shank3C, significantly increase the surface expression of the mouse hyperpolarization-activated, cyclic-nucleotide-gated (HCN) channel. In Shank3Δ13-16 knockout mice, which lack exons 13-16 in the Shank3 gene (both Shank3A and Shank3C are removed) and display a severe behavioural phenotype, the expression of HCN2 is reduced to an undetectable level. The thalamocortical (TC) neurons from the ventrobasal (VB) complex of Shank3Δ13-16 mice demonstrate reduced Ih current amplitude and correspondingly increased input resistance, negatively shifted resting membrane potential, and abnormal spike firing in both tonic and burst modes. Impressively, these changes closely resemble those of HCN2-/- TC neurons but not of the TC neurons from Shank3Δ4-9 mice, which lack exons 4-9 in the Shank3 gene (Shank3C still exists) and demonstrate moderate behavioural phenotypes. Additionally, Shank3 deficiency increases the ratio of excitatory/inhibitory balance in VB neurons but has a limited impact on the electrical properties of connected thalamic reticular (RTN) neurons. These results provide new understanding about the role of HCN channelopathy in mediating detrimental effects downstream from Shank3 deficiency.

Reduced GABAergic transmission in the ventrobasal thalamus contributes to thermal hyperalgesia in chronic inflammatory pain

The ventrobasal (VB) thalamus is innervated by GABAergic afferents from the thalamic reticular nucleus (TRN) and participates in nociception. But how the TRN-VB pathway regulates pain is not fully understood. In the present study, we reported decreased extracellular GABA levels in the VB of rats with CFA-induced chronic inflammatory pain, measured by microdialysis with HPLC analysis. In vitro whole-cell patch-clamp recording showed decreased amplitudes of tonic currents, increased frequencies of mIPSCs, and increased paired-pulse ratios in thalamic slices from chronic inflammatory rats (7 days). Microinjection of the GABAAR agonist muscimol and optogenetic activation of the TRN-VB pathway relieved thermal hyperalgesia in chronic inflammatory pain. By contrast, microinjecting the extrasynaptic GABAAR agonist THIP or selective knockout of synaptic GABAAR γ2 subunits aggravated thermal hyperalgesia in the chronic stage of inflammatory pain. Our findings indicate that reduced GABAergic transmission in the VB contributes to thermal hyperalgesia in chronic inflammatory pain, which could be a synaptic target for pharmacotherapy.

Differential alterations of intracellular [Ca2+] dynamics induced by cocaine and methylphenidate in thalamocortical ventrobasal neurons.

The ventrobasal (VB) thalamus relay nucleus processes information from rodents' whiskers, projecting to somatosensory cortex. Cocaine and methylphenidate (MPH) have been described to differentially alter intrinsic properties of, and spontaneous GABAergic input to, VB neurons. Here we studied using bis-fura 2 ratiometric fluorescence the effects of cocaine and MPH on intracellular [Ca2+] dynamics at the soma and dendrites of VB neurons. Cocaine increased baseline fluorescence in VB somatic and dendritic compartments. Peak and areas of fluorescence amplitudes were reduced by cocaine binge treatment in somas and dendrites at different holding potentials. MPH binge treatment did not alter ratiometric fluorescence at either somatic or dendritic levels. These novel cocaine-mediated blunting effects on intracellular [Ca2+] might account for alterations in the capacity of thalamocortical neurons to maintain gamma band oscillations, as well as their ability to integrate synaptic afferents.

A developmental study of glutamatergic neuron populations in the ventrobasal and the lateral geniculate nucleus of the thalamus: Comparing Genetic Absence Rats from Strasbourg (GAERS) and normal control wistar rats

An imbalance of GABAergic inhibition and glutamatergic excitation is suspected to be the cause of absence epileptic seizures. Absence seizures are known to be generated in thalamocortical circuitry. In the present study we used light microscopy immunohistochemistry to quantify the density of glutamate+ve neurons at two developmental stages (P10 and P60) in two thalamic nuclei, the ventrobasal (VB) and lateral geniculate nucleus (LGN) in Wistar rats and compared the results with similar data obtained from genetic absence epilepsy rats from Strasbourg (GAERS).Rats were perfused transcardially with glutaraldehyde and paraformaldehyde fixative, then samples from VB and LGN were removed from each animal and sectioned. The glutamatergic neurons were labelled using light-microscopic glutamate immunohistochemistry. The disector method was used to quantify the glutamate+ve neurons in VB and LGN of GAERS and Wistar rats. The data were statistically analyzed.The distribution of the glutamate+ve neurons in the VB thalamic nucleus showed a significant reduction in the neuronal profiles per unit thalamic area from P10 to P60 in both Wistar and GAERS. The decrease was greater in the GAERS compared to the Wistar animals. However, in the LGN no reduction was observed either in the Wistar or in the GAERS. Comparing the density of glutamate+ve neurons in the VB thalamic nucleus of P10 of Wistar animals with of P10 GAERS showed statistically significant greater densities of these neurons in GAERS than in the Wistar rats. However no significant difference was present at P60 between the Wistar and GAERS animals.The disproportional decrease in GAERS may be related to the onset of absence seizures or may be related to neurogenesis of absence epilepsy.

Electrical synapses and the development of inhibitory circuits in the thalamus.

The thalamus is a structure critical for information processing and transfer to the cortex. Thalamic reticular neurons are inhibitory cells interconnected by electrical synapses, most of which require the gap junction protein connexin36 (Cx36). We investigated whether electrical synapses play a role in the maturation of thalamic networks by studying neurons in mice with and without Cx36. When Cx36 was deleted, inhibitory synapses were more numerous, although both divergent inhibitory connectivity and dendritic complexity were reduced. Surprisingly, we observed non-Cx36-dependent electrical synapses with unusual biophysical properties interconnecting some reticular neurons in mice lacking Cx36. The results of the present study suggest an important role for Cx36-dependent electrical synapses in the development of thalamic circuits. Neurons within the mature thalamic reticular nucleus (TRN) powerfully inhibit ventrobasal (VB) thalamic relay neurons via GABAergic synapses. TRN neurons are also coupled to one another by electrical synapses that depend strongly on the gap junction protein connexin36 (Cx36). Electrical synapses in the TRN precede the postnatal development of TRN-to-VB inhibition. We investigated how the deletion of Cx36 affects the maturation of TRN and VB neurons, electrical coupling and GABAergic synapses by studying wild-type (WT) and Cx36 knockout (KO) mice. The incidence and strength of electrical coupling in TRN was sharply reduced, but not abolished, in KO mice. Surprisingly, electrical synapses between Cx36-KO neurons had faster voltage-dependent decay kinetics and conductance asymmetry (rectification) than did electrical synapses between WT neurons. The properties of TRN-mediated inhibition in VB also depended on the Cx36 genotype. Deletion of Cx36 increased the frequency and shifted the amplitude distributions of miniature IPSCs, whereas the paired-pulse ratio of evoked IPSCs was unaffected, suggesting that the absence of Cx36 led to an increase in GABAergic synaptic contacts. VB neurons from Cx36-KO mice also tended to have simpler dendritic trees and fewer divergent inputs from the TRN compared to WT cells. The findings obtained in the present study suggest that proper development of thalamic inhibitory circuitry, neuronal morphology, TRN cell function and electrical coupling requires Cx36. In the absence of Cx36, some TRN neurons express asymmetric electrical coupling mediated by other unidentified connexin subtypes.

Anti-absence activity of mGlu1 and mGlu5 receptor enhancers and their interaction with a GABA reuptake inhibitor: Effect of local infusions in the somatosensory cortex and thalamus.

Glutamate and γ-aminobutyric acid (GABA) are the key neurotransmitter systems in the cortical-thalamocortical network, involved in normal and pathologic oscillations such as spike-wave discharges (SWDs), which characterize different forms of absence epilepsy. Metabotropic glutamate (mGlu) and GABA receptors are widely expressed within this network. Herein, we examined the effects of two selective positive allosteric modulators (PAMs) of mGlu1 and mGlu5 receptors, the GABA reuptake inhibitor, tiagabine, and their interaction in the somatosensory cortex and thalamus on SWDs in WAG/Rij rats. Male WAG/Rij rats were equipped with bilateral cannulas in the somatosensory cortex (S1po) or the ventrobasal (VB) thalamic nuclei, and with cortical electroencephalography (EEG) electrodes. Rats received a single dose of the mGlu1 receptor PAM, RO0711401, or the mGlu5 receptor PAM, VU0360172, various doses of tiagabine, or VU0360172 combined with tiagabine. Both PAMs suppressed SWDs regardless of the site of injection. Tiagabine enhanced SWDs when injected into the thalamus, but, unexpectedly, suppressed SWDs in a dose-dependent manner when injected into the cortex. Intracortical co-injection of VU0360172 and tiagabine produced slightly larger effects as compared to either VU0360172 or tiagabine alone. Intrathalamic co-injections of VU0360172 and subthreshold doses of tiagabine caused an antiabsence effect similar to that exhibited by VU0360172 alone in the first 10 min. At 30 min, however, the antiabsence effect of VU0360172 was prevented by subthreshold doses of tiagabine, and the combination produced a paradoxical proabsence effect at 40 and 50 min. These data (1) show that mGlu1 and mGlu5 receptor PAMs reduce absence seizures acting at both thalamic and cortical levels; (2) demonstrate for the first time that tiagabine, despite its established absence-enhancing effect, reduces SWDs when injected into the somatosensory cortex; and (3) indicate that the efficacy of VU0360172 in the thalamus may be critically affected by the availability of (extra)synaptic GABA.

Developmentally regulated neurosteroid synthesis enhances GABAergic neurotransmission in mouse thalamocortical neurones.

During neuronal development synaptic events mediated by GABAA receptors are progressively reduced in their duration, allowing for rapid and precise network function. Here we focused on ventrobasal thalamocortical neurones, which contribute to behaviourally relevant oscillations between thalamus and cortex. We demonstrate that the developmental decrease in the duration of inhibitory phasic events results predominantly from a precisely timed loss of locally produced neurosteroids, which act as positive allosteric modulators of the GABAA receptor. The mature thalamus retains the ability to synthesise neurosteroids, thus preserving the capacity to enhance both phasic and tonic inhibition, mediated by synaptic and extrasynaptic GABAA receptors, respectively, in physiological and pathophysiological scenarios associated with perturbed neurosteroid levels. Our data establish a potent, endogenous mechanism to locally regulate the GABAA receptor function and thereby influence thalamocortical activity. During brain development the duration of miniature inhibitory postsynaptic currents (mIPSCs) mediated by GABAA receptors (GABAA Rs) progressively reduces, to accommodate the temporal demands required for precise network activity. Conventionally, this synaptic plasticity results from GABAA R subunit reorganisation. In particular, in certain developing neurones synaptic α2-GABAA Rs are replaced by α1-GABAA Rs. However, in thalamocortical neurones of the mouse ventrobasal (VB) thalamus, the major alteration to mIPSC kinetics occurs on postnatal (P) day 10, some days prior to the GABAA R isoform change. Here, whole-cell voltage-clamp recordings from VB neurones of mouse thalamic slices revealed that early in postnatal development (P7-P8), the mIPSC duration is prolonged by local neurosteroids acting in a paracrine or autocrine manner to enhance GABAA R function. However, by P10, this neurosteroid 'tone' rapidly dissipates, thereby producing brief mIPSCs. This plasticity results from a lack of steroid substrate as pre-treatment of mature thalamic slices (P20-24) with the GABAA R-inactive precursor 5α-dihydroprogesterone (5α-DHP) resulted in markedly prolonged mIPSCs and a greatly enhanced tonic conductance, mediated by synaptic and extrasynaptic GABAA Rs, respectively. In summary, endogenous neurosteroids profoundly influence GABAergic neurotransmission in developing VB neurones and govern a transition from slow to fast phasic synaptic events. Furthermore, the retained capacity for steroidogenesis in the mature thalamus raises the prospect that certain physiological or pathophysiological conditions may trigger neurosteroid neosynthesis, thereby providing a local mechanism for fine-tuning neuronal excitability.

Altered intrathalamic GABAA neurotransmission in a mouse model of a human genetic absence epilepsy syndrome

We previously demonstrated that heterozygous deletion of Gabra1, the mouse homolog of the human absence epilepsy gene that encodes the GABAA receptor (GABAAR) α1 subunit, causes absence seizures. We showed that cortex partially compensates for this deletion by increasing the cell surface expression of residual α1 subunit and by increasing α3 subunit expression. Absence seizures also involve two thalamic nuclei: the ventrobasal (VB) nucleus, which expresses only the α1 and α4 subtypes of GABAAR α subunits, and the reticular (nRT) nucleus, which expresses only the α3 subunit subtype. Here, we found that, unlike cortex, VB exhibited significantly reduced total and synaptic α1 subunit expression. In addition, heterozygous α1 subunit deletion substantially reduced miniature inhibitory postsynaptic current (mIPSC) peak amplitudes and frequency in VB. However, there was no change in the expression of the extrasynaptic α4 or δ subunits in VB and, unlike other models of absence epilepsy, no change in tonic GABAAR currents. Although heterozygous α1 subunit knockout increased α3 subunit expression in medial thalamic nuclei, it did not alter α3 subunit expression in nRT. However, it did enlarge the presynaptic vesicular inhibitory amino acid transporter puncta and lengthen the time constant of mIPSC decay in nRT. We conclude that increased tonic GABAA currents are not necessary for absence seizures. In addition, heterozygous loss of α1 subunit disinhibits VB by substantially reducing phasic GABAergic currents and surprisingly, it also increases nRT inhibition by prolonging phasic currents. The increased inhibition in nRT likely represents a partial compensation that helps reduce absence seizures.

GABA B receptor-mediated activation of astrocytes by gamma-hydroxybutyric acid

The gamma-aminobutyric acid (GABA) metabolite gamma-hydroxybutyric acid (GHB) shows a variety of behavioural effects when administered to animals and humans, including reward/addiction properties and absence seizures. At the cellular level, these actions of GHB are mediated by activation of neuronal GABAB receptors (GABABRs) where it acts as a weak agonist. Because astrocytes respond to endogenous and exogenously applied GABA by activation of both GABAA and GABABRs, here we investigated the action of GHB on astrocytes on the ventral tegmental area (VTA) and the ventrobasal (VB) thalamic nucleus, two brain areas involved in the reward and proepileptic action of GHB, respectively, and compared it with that of the potent GABABR agonist baclofen. We found that GHB and baclofen elicited dose-dependent (ED50: 1.6 mM and 1.3 µM, respectively) transient increases in intracellular Ca2+ in VTA and VB astrocytes of young mice and rats, which were accounted for by activation of their GABABRs and mediated by Ca2+ release from intracellular store release. In contrast, prolonged GHB and baclofen exposure caused a reduction in spontaneous astrocyte activity and glutamate release from VTA astrocytes. These findings have key (patho)physiological implications for our understanding of the addictive and proepileptic actions of GHB.

Thalamocortical neurons display suppressed burst-firing due to an enhanced Ih current in a genetic model of absence epilepsy.

Burst-firing in distinct subsets of thalamic relay (TR) neurons is thought to be a key requirement for the propagation of absence seizures. However, in the well-regarded Genetic Absence Epilepsy Rats from Strasbourg (GAERS) model as yet there has been no link described between burst-firing in TR neurons and spike-and-wave discharges (SWDs). GAERS ventrobasal (VB) neurons are a specific subset of TR neurons that do not normally display burst-firing during absence seizures in the GAERS model, and here, we assessed the underlying relationship of VB burst-firing with Ih and T-type calcium currents between GAERS and non-epileptic control (NEC) animals. In response to 200-ms hyperpolarizing current injections, adult epileptic but not pre-epileptic GAERS VB neurons displayed suppressed burst-firing compared to NEC. In response to longer duration 1,000-ms hyperpolarizing current injections, both pre-epileptic and epileptic GAERS VB neurons required significantly more hyperpolarizing current injection to burst-fire than those of NEC animals. The current density of the Hyperpolarization and Cyclic Nucleotide-activated (HCN) current (Ih) was found to be increased in GAERS VB neurons, and the blockade of Ih relieved the suppressed burst-firing in both pre-epileptic P15–P20 and adult animals. In support, levels of HCN-1 and HCN-3 isoform channel proteins were increased in GAERS VB thalamic tissue. T-type calcium channel whole-cell currents were found to be decreased in P7–P9 GAERS VB neurons, and also noted was a decrease in CaV3.1 mRNA and protein levels in adults. Z944, a potent T-type calcium channel blocker with anti-epileptic properties, completely abolished hyperpolarization-induced VB burst-firing in both NEC and GAERS VB neurons.

γ-Hydroxybutyric Acid (GHB) Is Not an Agonist of Extrasynaptic GABAA Receptors

γ-Hydroxybutyric acid (GHB) is an endogenous compound and a drug used clinically to treat the symptoms of narcolepsy. GHB is known to be an agonist of GABAB receptors with millimolar affinity, but also binds with much higher affinity to another site, known as the GHB receptor. While a body of evidence has shown that GHB does not bind to GABAA receptors widely, recent evidence has suggested that the GHB receptor is in fact on extrasynaptic α4β1δ GABAA receptors, where GHB acts as an agonist with an EC50 of 140 nM. We investigated three neuronal cell types that express a tonic GABAA receptor current mediated by extrasynaptic receptors: ventrobasal (VB) thalamic neurons, dentate gyrus granule cells and striatal medium spiny neurons. Using whole-cell voltage clamp in brain slices, we found no evidence that GHB (10 µM) induced any GABAA receptor mediated current in these cell types, nor that it modulated inhibitory synaptic currents. Furthermore, a high concentration of GHB (3 mM) was able to produce a GABAB receptor mediated current, but did not induce any other currents. These results suggest either that GHB is not a high affinity agonist at native α4β1δ receptors, or that these receptors do not exist in classical areas associated with extrasynaptic currents.