Sex differences in motor learning flexibility are accompanied by sex differences in mushroom spine pruning of the mouse primary motor cortex during adolescence.

Background: Although males excel at motor tasks requiring strength, females exhibit greater motor learning flexibility. Cognitive flexibility is associated with low baseline mushroom spine densities achieved by pruning which can be triggered by α4ßδ GABAA receptors (GABARs); defective synaptic pruning impairs this process. Methods: We investigated sex differences in adolescent pruning of mushroom spine pruning of layer 5 pyramidal cells of primary motor cortex (L5M1), a site essential for motor learning, using microscopic evaluation of Golgi stained sections. We assessed α4GABAR expression using immunohistochemical and electrophysiological techniques (whole cell patch clamp responses to 100 nM gaboxadol, selective for α4ßδ GABARs). We then compared performance of groups with different post-pubertal mushroom spine densities on motor learning (constant speed) and learning flexibility (accelerating speed following constant speed) rotarod tasks. Results: Mushroom spines in proximal L5M1 of female mice decreased >60% from PND35 (puberty onset) to PND56 (Pubertal: 2.23 ± 0.21 spines/10 µm; post-pubertal: 0.81 ± 0.14 spines/10 µm, P < 0.001); male mushroom spine density was unchanged. This was due to greater α4ßδ GABAR expression in the female (P < 0.0001) because α4 -/- mice did not exhibit mushroom spine pruning. Although motor learning was similar for all groups, only female wild-type mice (low mushroom spine density) learned the accelerating rotarod task after the constant speed task (P = 0.006), a measure of motor learning flexibility. Conclusions: These results suggest that optimal motor learning flexibility of female mice is associated with low baseline levels of post-pubertal mushroom spine density in L5M1 compared to male and female α4 -/- mice.

Manipulation of α4ßδ GABAA receptors alters synaptic pruning in layer 3 prelimbic prefrontal cortex and impairs temporal order recognition: Implications for schizophrenia and autism.

Temporal order memory is impaired in autism spectrum disorder (ASD) and schizophrenia (SCZ). These disorders, more prevalent in males, result in abnormal dendritic spine pruning during adolescence in layer 3 (L3) medial prefrontal cortex (mPFC), yielding either too many (ASD) or too few (SCZ) spines. Here we tested whether altering spine density in neural circuits including the mPFC could be associated with impaired temporal order memory in male mice. We have shown that α4ßδ GABAA receptors (GABARs) emerge at puberty on spines of L5 prelimbic mPFC (PL) where they trigger pruning. We show here that α4ßδ receptors also increase at puberty in L3 PL (P < 0.0001) and used these receptors as a target to manipulate spine density here. Pubertal injection (14 d) of the GABA agonist gaboxadol, at a dose (3 mg/kg) selective for α4ßδ, reduced L3 spine density by half (P < 0.0001), while α4 knock-out increased spine density âˆ¼ 40 % (P < 0.0001), mimicking spine densities in SCZ and ASD, respectively. In both cases, performance on the mPFC-dependent temporal order recognition task was impaired, resulting in decreases in the discrimination ratio which assesses preference for the novel object: -0.39 ± 0.15, gaboxadol versus 0.52 ± 0.09, vehicle; P = 0.0002; -0.048 ± 0.10, α4 KO versus 0.49 ± 0.04, wild-type; P < 0.0001. In contrast, the number of approaches was unaltered, reflecting unchanged locomotion. These data suggest that altering α4ßδ GABAR expression/activity alters spine density in L3 mPFC and impairs temporal order memory to mimic changes in ASD and SCZ. These findings may provide insight into these disorders.

Preventing adolescent synaptic pruning in mouse prelimbic cortex via local knockdown of α4ßδ GABAA receptors increases anxiety response in adulthood.

Anxiety is increasingly reported, especially in adolescent females. The etiology is largely unknown, which limits effective treatment. Layer 5 prelimbic cortex (L5PL) increases anxiety responses but undergoes adolescent synaptic pruning, raising the question of the impact of pruning on anxiety. Here we show that preventing L5PL pruning increases anxiety in response to an aversive event in adolescent and adult female mice. Spine density of Golgi-stained neurons decreased ~ 63% from puberty (~ PND35, vaginal opening) to post-puberty (PND56, P < 0.0001). Expression of α4ßδ GABAA receptors (GABARs) transiently increased tenfold in L5PL at puberty (P < 0.00001), but decreased post-pubertally. Both global and local knockdown of these receptors during puberty prevented pruning, increasing spine density post-pubertally (P < 0.0001), an effect reversed by blocking NMDA receptors (NMDARs). Pubertal expression of the NMDAR-dependent spine protein kalirin7 decreased (50%, P < 0.0001), an effect prevented by α4 knock-out, suggesting that α4ßδ-induced reductions in kalirin7 underlie pruning. Increased spine density due to local α4 knockdown at puberty decreased open arm time on the elevated plus maze post-pubertally (62%, P < 0.0001) in response to an aversive stimulus, suggesting that increases in L5PL synapses increase anxiety responses. These findings suggest that prelimbic synaptic pruning is necessary to limit anxiety in adulthood and may suggest novel therapies.

Increased Dendritic Branching of and Reduced δ-GABAA Receptor Expression on Parvalbumin-Positive Interneurons Increase Inhibitory Currents and Reduce Synaptic Plasticity at Puberty in Female Mouse CA1 Hippocampus.

Parvalbumin positive (PV+) interneurons play a pivotal role in cognition and are known to be regulated developmentally and by ovarian hormones. The onset of puberty represents the end of a period of optimal learning when impairments in synaptic plasticity are observed in the CA1 hippocampus of female mice. Therefore, we tested whether the synaptic inhibitory current generated by PV+ interneurons is increased at puberty and contributes to these deficits in synaptic plasticity. To this end, the spontaneous inhibitory postsynaptic current (sIPSC) was recorded using whole-cell patch-clamp techniques from CA1 pyramidal cells in the hippocampal slice before (PND 28-32) and after the onset of puberty in female mice (~PND 35-44, assessed by vaginal opening). sIPSC frequency and amplitude were significantly increased at puberty, but these measures were reduced by 1 µM DAMGO [1 µM, (D-Ala2, N-MePhe4, Gly-ol)-enkephalin], which silences PV+ activity via µ-opioid receptor targets. At puberty, dendritic branching of PV+ interneurons in GAD67-GFP mice was increased, while expression of the δ subunit of the GABAA receptor (GABAR) on these interneurons decreased. Both frequency and amplitude of sIPSCs were significantly increased in pre-pubertal mice with reduced δ expression, suggesting a possible mechanism. Theta burst induction of long-term potentiation (LTP), an in vitro model of learning, is impaired at puberty but was restored to optimal levels by DAMGO administration, implicating inhibition via PV+ interneurons as one cause. Administration of the neurosteroid/stress steroid THP (30 nM, 3α-OH, 5α-pregnan-20-one) had no effect on sIPSCs. These findings suggest that phasic inhibition generated by PV+ interneurons is increased at puberty when it contributes to impairments in synaptic plasticity. These results may have relevance for the changes in cognitive function reported during early adolescence.

Pubertal hormones increase hippocampal expression of α4ßδ GABAA receptors.

CA1 hippocampal expression of α4ßδ GABAA receptors (GABARs) increases at the onset of puberty in female mice, an effect dependent upon the decline in hippocampal levels of the neurosteroid THP (3α-OH-5α-pregnan-20-one) which occurs at this time. The present study further characterized the mechanisms underlying α4ßδ expression, assessed in vivo. Blockade of pubertal levels of 17ß-estradiol (E2) (formestane, 0.5 mg/kg, i.p. 3 d) reduced α4 and δ expression by 75-80% (P < 0.05) in CA1 hippocampus of female mice, assessed using Western blot techniques. Conversely, E2 administration increased α4 and δ expression by 50-100% in adults, an effect enhanced by more than 2-fold by concomitant administration of the 5α-reductase blocker finasteride (50 mg/kg, i.p., 3d, P < 0.05), suggesting that both declining THP levels and increasing E2 levels before puberty trigger α4ßδ expression. This effect was blocked by ICI 182,780 (20 mg/kg, s.c., 3 d), a selective blocker of E2 receptor-α (ER-α). These results suggest that both the rise in circulating levels of E2 and the decline in hippocampal THP levels at the onset of puberty trigger maximal levels of α4ßδ expression in the CA1 hippocampus.

α4ßδ GABAA Receptors Trigger Synaptic Pruning and Reduce Dendritic Length of Female Mouse CA3 Hippocampal Pyramidal Cells at Puberty.

Synaptic pruning during adolescence is critical for optimal cognition. The CA3 hippocampus contains unique spine types and plays a pivotal role in pattern separation and seizure generation, where sex differences exist, but adolescent pruning has only been studied in the male. Thus, for the present study we assessed pruning of specific spine types in the CA3 hippocampus during adolescence and investigated a possible mechanism in the female mouse. To this end, we used Golgi-impregnated brains from pubertal (∼PND 35, assessed by vaginal opening) and post-pubertal (PND 56) mice. Spine density was assessed from z-stack (0.1-µm steps) images taken using a Nikon DS-U3 camera through a Nikon Eclipse Ci-L microscope and analyzed with NIS Elements. Spine density decreased significantly (P < 0.05) during adolescence, with 50-60% decreases in mushroom and stubby spine-types (P < 0.05, ∼PND35 vs. PND56) in non-proestrous mice. This was associated with decreases in kalirin-7, a spine protein which stabilizes the cytoskeleton and is required for spine maintenance. Because our previous findings suggest that pubertal increases in α4ßδ GABAA receptors (GABARs) trigger pruning in CA1, we investigated their role in CA3. α4 expression in CA3 hippocampus increased 4-fold at puberty (P < 0.05), assessed by immunostaining and verified electrophysiologically by an increased response to gaboxadol (100 nM), which is selective for α4ßδ. Knock-out of α4 prevented the pubertal decrease in kalirin-7 and synaptic pruning and also increased the dendritic length, demonstrating a functional link. These data suggest that pubertal α4ßδ GABARs alter dendritic morphology and trigger pruning in female CA3 hippocampus.

α4ßδ GABAA receptors reduce dendritic spine density in CA1 hippocampus and impair relearning ability of adolescent female mice: Effects of a GABA agonist and a stress steroid.

Synaptic pruning underlies the transition from an immature to an adult CNS through refinements of neuronal circuits. Our recent study indicates that pubertal synaptic pruning is triggered by the inhibition generated by extrasynaptic α4ßδ GABAA receptors (GABARs) which are increased for 10 d on dendritic spines of CA1 pyramidal cells at the onset of puberty (PND 35-44) in the female mouse, suggesting α4ßδ GABARs as a novel target for the regulation of adolescent synaptic pruning. In the present study we used a pharmacological approach to further examine the role of these receptors in altering spine density during puberty of female mice and the impact of these changes on spatial learning, assessed in adulthood. Two drugs were chronically administered during the pubertal period (PND 35-44): the GABA agonist gaboxadol (GBX, 0.1mg/kg, i.p.), to enhance current gated by α4ßδ GABARs and the neurosteroid/stress steroid THP (3α-OH-5ß-pregnan-20-one, 10mg/kg, i.p.) to decrease expression of α4ßδ. Spine density was determined on PND 56 with Golgi staining. Spatial learning and relearning were assessed using the multiple object relocation task and an active place avoidance task on PND 56. Pubertal GBX decreased spine density post-pubertally by 70% (P<0.05), while decreasing α4ßδ expression with THP increased spine density by twofold (P<0.05), in both cases, with greatest effects on the mushroom spines. Adult relearning ability was compromised in both hippocampus-dependent tasks after pubertal administration of either drug. These findings suggest that an optimal spine density produced by α4ßδ GABARs is necessary for optimal cognition in adults.

Role of α4-containing GABAA receptors in limiting synaptic plasticity and spatial learning of female mice during the pubertal period.

Expression of α4ßδ GABAA receptors (GABARs) increases at the onset of puberty on dendritic spines of CA1 hippocampal pyramidal cells. These receptors reduce activation of NMDA receptors (NMDARs), impair induction of long-term potentiation (LTP) and reduce hippocampal-dependent spatial learning. These effects are not seen in the δ-/- mouse, implicating α4ßδ GABARs. Here we show that knock-out of α4 also restores synaptic plasticity and spatial learning in female mice at the onset of puberty (verified by vaginal opening). To this end, field excitatory post-synaptic potentials (fEPSPs) were recorded from the stratum radiatum of CA1 hippocampus in the slice from +/+ and α4-/- pubertal mice (PND 35-44). Induction of LTP, in response to stimulation of the Schaffer collaterals with theta burst stimulation (TBS), was unsuccessful in the +/+ hippocampus, but reinstated by α4 knock-out (~65% potentiation) but not by blockade of α5-GABARs with L-655,708 (50nM). In order to compare spatial learning in the two groups of mice, animals were trained in an active place avoidance task where the latency to first enter a shock zone is a measure of learning. α4-/- mice had significantly longer latencies by the third learning trial, suggesting better spatial learning, compared to +/+ animals, who did not reach the criterion for learning (120s latency). These findings suggest that knock-out of the GABAR α4 subunit restores synaptic plasticity and spatial learning at puberty and is consistent with the concept that the dendritic α4ßδ GABARs which emerge at puberty selectively impair CNS plasticity. This article is part of a Special Issue entitled SI: Adolescent plasticity.

Pubertal Expression of α4ßδ GABAA Receptors Reduces Seizure-Like Discharges in CA1 Hippocampus.

More than half of children with epilepsy outgrow their seizures, yet the underlying mechanism is unknown. GABAergic inhibition increases at puberty in female mice due to expression of extrasynaptic α4ßδ GABAA receptors (GABARs). Therefore, we tested the role of these receptors in regulating seizure-like discharges in CA1 hippocampus using a high K(+) (8.5 mM) seizure model. Spontaneous field potentials were recorded from hippocampus of pre-pubertal (~28-32 PND) and pubertal (~35-44 PND) female wild-type or α4-/- mice. The coastline length, a measure of burst intensity, was assessed. 8.5 mM K(+) induced seizure-like discharges in over 60% of pre-pubertal slices, but only in 7% of pubertal slices, where the coastline length was reduced by 70% (P = 0.04). However, the pubertal decrease in seizure-like discharges was not seen in the α4-/-, implicating α4ßδ GABARs as the cause of the decreased seizure-like activity during puberty. Administration of THIP or DS2, to selectively increase α4ßδ current, reduced activity in 8.5 mM K(+) at puberty, while blockade of α5-GABARs had no effect. GABAergic current was depolarizing but inhibitory in 8.5 mM K(+), suggesting a mechanism for the effects of α4ßδ and α5-GABARs, which exhibit different polarity-dependent desensitization. These data suggest that α4ßδ GABARs are anti-convulsant during adolescence.

Flumazenil decreases surface expression of α4ß2δ GABAA receptors by increasing the rate of receptor internalization.

Increases in expression of α4ßδ GABAA receptors (GABARs), triggered by fluctuations in the neurosteroid THP (3α-OH-5α[ß]-pregnan-20-one), are associated with changes in mood and cognition. We tested whether α4ßδ trafficking and surface expression would be altered by in vitro exposure to flumazenil, a benzodiazepine ligand which reduces α4ßδ expression in vivo. We first determined that flumazenil (100 nM-100 µM, IC50=∼1 µM) acted as a negative modulator, reducing GABA (10 µM)-gated current in the presence of 100 nM THP (to increase receptor efficacy), assessed with whole cell patch clamp recordings of recombinant α4ß2δ expressed in HEK-293 cells. Surface expression of recombinant α4ß2δ receptors was detected using a 3XFLAG reporter at the C-terminus of α4 (α4F) using confocal immunocytochemical techniques following 48 h exposure of cells to GABA (10 µM)+THP (100 nM). Flumazenil (10 µM) decreased surface expression of α4F by ∼60%, while increasing its intracellular accumulation, after 48 h. Reduced surface expression of α4ß2δ after flumazenil treatment was confirmed by decreases in the current responses to 100 nM of the GABA agonist gaboxadol. Flumazenil-induced decreases in surface expression of α4ß2δ were prevented by the dynamin blocker, dynasore, and by leupeptin, which blocks lysosomal enzymes, suggesting that flumazenil is acting to increase endocytosis and lysosomal degradation of the receptor. Flumazenil increased the rate of receptor removal from the cell surface by 2-fold, assessed using botulinum toxin B to block insertion of new receptors. These findings may suggest new therapeutic strategies for regulation of α4ß2δ expression using flumazenil.

Neurosteroid effects at α4ßδ GABAA receptors alter spatial learning and synaptic plasticity in CA1 hippocampus across the estrous cycle of the mouse.

Fluctuations in circulating levels of ovarian hormones have been shown to regulate cognition (Sherwin and Grigorova, 2011. Fertil. Steril. 96, 399-403; Shumaker et al., 2004. JAMA. 291, 2947-2958), but increases in estradiol on the day of proestrus yield diverse outcomes: In vivo induction of long-term potentiation (LTP), a model of learning, is reduced in the morning, but optimal in the afternoon (Warren et al., 1995. Brain Res. 703, 26-30). The mechanism underlying this discrepancy is not known. Here, we show that impairments in both CA1 hippocampal LTP and spatial learning observed on the morning of proestrus are due to increased dendritic expression of α4ßδ GABAA receptors (GABARs) on CA1 pyramidal cells, as assessed by electron microscopic (EM) techniques, compared with estrus and diestrus. LTP induction and spatial learning were robust, however, when assessed on the morning of proestrus in α4-/- mice, implicating these receptors in mediating impaired plasticity. Although α4ßδ expression remained elevated on the afternoon of proestrus, increases in 3α-OH-THP (3α-OH-5α-pregnan-20-one) decreased inhibition by reducing outward current through α4ßδ GABARs (Shen et al., 2007. Nat. Neurosci. 10, 469-477), in contrast to the usual effect of this steroid to enhance inhibition. Proestrous levels of 3α-OH-THP reversed the deficits in LTP and spatial learning, an effect prevented by the inactive metabolite 3ß-OH-THP (10 mg/kg, i.p.), which antagonizes actions of 3α-OH-THP. In contrast, administration of 3α-OH-THP (10 mg/kg, i.p.) on the morning of proestrus improved spatial learning scores 150-300%. These findings suggest that cyclic fluctuations in ovarian steroids can induce changes in cognition via α4ßδ GABARs that are dependent upon 3α-OH-THP. This article is part of a Special Issue entitled SI: Brain and Memory.

Characterization of neurosteroid effects on hyperpolarizing current at α4ß2δ GABAA receptors.

RATIONALE: The neurosteroid 3α,5ß-THP (3α-OH-5ß-pregnan-20-one, pregnanolone) is a modulator of the GABAA receptor (GABAR), with α4ß2δ GABARs the most sensitive. However, the effects of 3α,5ß-THP at α4ß2δ are polarity-dependent: 3α,5ß-THP potentiates depolarizing current, as has been widely reported, but decreases hyperpolarizing current by accelerating desensitization. OBJECTIVES: The present study further characterized 3α,5ß-THP inhibition of hyperpolarizing current at this receptor and compared effects of other related steroids at α4ß2δ GABARs. METHODS: α4ß2δ GABARs were expressed in HEK-293 cells, and agonist-gated current recorded with whole cell voltage-clamp techniques using a theta tube to rapidly apply agonist before and after application of neurosteroids. RESULTS: The GABA-modulatory steroids (30 nM) 3α,5α-THP (3α-OH-5α-pregnan-20-one, allopregnanolone) and THDOC (3α,21-dihydroxy-5α-pregnan-20-one) inhibited hyperpolarizing GABA (10 µM)-gated current at α4ß2δ GABARs similar to 3α,5ß-THP, while the inactive 3ß,5ß-THP isomer had no effect. Greater inhibition was seen for current gated by the high efficacy agonist gaboxadol (THIP, 100 µM) than for GABA (0.1-1000 µM), consistent with an effect of 3α,5ß-THP on desensitization. Inhibitory effects of the steroid were not seen under low [Cl(-)] conditions or in the presence of calphostin C (500 nM), an inhibitor of protein kinase C. Chimeras swapping the IL (intracellular loop) of α4 with α1, when expressed with ß2 and δ, produced receptors (α[414]ß2δ) which were not inhibited by 3α,5ß-THP when GABA-gated current was hyperpolarizing, while α[141]ß2δ exhibited steroid-induced polarity-dependent modulation. CONCLUSIONS: These findings suggest that numerous neurosteroids exhibit polarity-dependent effects at α4ß2δ GABARs, which are dependent upon protein kinase C and the IL of α4.

α4ßδ GABAA receptors and tonic inhibitory current during adolescence: effects on mood and synaptic plasticity.

The onset of puberty is associated with alterations in mood as well as changes in cognitive function, which can be more pronounced in females. Puberty onset in female mice is associated with increased expression of α4ßδ γ-amino-butyric acid-A (GABAA) receptors (GABARs) in CA1 hippocampus. These receptors, which normally have low expression in this central nervous system (CNS) site, emerge along the apical dendrites as well as on the dendritic spines of pyramidal neurons, adjacent to excitatory synapses where they underlie a tonic inhibition that shunts excitatory current and impairs activation of N-methyl-D-aspartate (NMDA) receptors, the trigger for synaptic plasticity. As would be expected, α4ßδ expression at puberty also prevents long-term potentiation (LTP), an in vitro model of learning which is a function of network activity, induced by theta burst stimulation of the Schaffer collaterals to the CA1 hippocampus. The expression of these receptors also impairs spatial learning in a hippocampal-dependent task. These impairments are not seen in δ knock-out (-/-) mice, implicating α4ßδ GABARs. α4ßδ GABARs are also a sensitive target for steroids such as THP ([allo]pregnanolone or 3α-OH-5α[ß]-pregnan-20-one), which are dependent upon the polarity of GABAergic current. It is well-known that THP can increase depolarizing current gated by α4ßδ GABARs, but more recent data suggest that THP can reduce hyperpolarizing current by accelerating receptor desensitization. At puberty, THP reduces the hyperpolarizing GABAergic current, which removes the shunting inhibition that impairs synaptic plasticity and learning at this time. However, THP, a stress steroid, also increases anxiety, via its action at α4ßδ GABARs because it is not seen in δ(-/-) mice. These findings will be discussed as well as their relevance to changes in mood and cognition at puberty, which can be a critical period for certain types of learning and when anxiety disorders and mood swings can emerge.

Regulation of the surface expression of α4ß2δ GABAA receptors by high efficacy states.

α4ßδ GABA(A) receptors (GABARs) have low CNS expression, but their expression is increased by 48h exposure to the neurosteroid THP (3α-OH-5α[ß]-pregnan-20-one). THP also increases the efficacy of δ-containing GABARs acutely, where GABA is a partial agonist. Thus, we examined effects of THP (100 nM) and full GABA agonists at α4ß2δ (gaboxadol, 10 µM, and ß-alanine, 10 µM-1mM), on surface expression of α4ß2δ. To this end, we used an α4 construct tagged with a 3XFLAG (F) epitope or measured expression of native α4 and δ. HEK-293 cells or cultured hippocampal neurons were transfected with α4Fß2δ and treated 24h later with GABA agonists, THP, GABA plus THP or vehicle (0.01% DMSO) for 0.5 h-48 h. Immunocytochemistry was performed under both non-permeabilized and permeabilized conditions to detect surface and intracellular labeling, respectively, using confocal microscopy. The high efficacy agonists and GABA (1 or 10 µM) plus THP increased α4ß2δ surface expression up to 3-fold after 48h, an effect first seen by 0.5h. This effect was not dependent upon the polarity of GABAergic current, although expression was increased by KCC2. Intracellular labeling was decreased while functional expression was confirmed by whole cell patch clamp recordings of responses to GABA agonists. GABA plus THP treatment did not alter the rate of receptor removal from the surface membrane, suggesting that THP-induced α4ß2δ expression is likely via receptor insertion. Surface expression of α4ß2δ was decreased by rottlerin (10 µM), suggesting a role for PKC-δ. These results suggest that trafficking of α4ß2δ GABARs is regulated by high efficacy states.

Knockout of the γ-aminobutyric acid receptor subunit α4 reduces functional δ-containing extrasynaptic receptors in hippocampal pyramidal cells at the onset of puberty.

Increased plasmalemmal localization of α4ßδ GABA(A) receptors (GABARs) occurs in the hippocampal pyramidal cells of female mice at pubertal onset (Shen et al., 2010). This increase occurs on both dendritic spines and shafts of CA1 pyramidal cells and is in response to hormone fluctuations that occur at pubertal onset. However, little is known about how the α4 and δ subunits individually mediate the formation of functional, plasmalemmal α4ßδ GABARs. To determine whether expression of the α4 subunit is necessary for plasmalemmal δ subunit localization at pubertal onset, electron microscopic-immunocytochemistry (EM-ICC) was employed. CA1 pyramidal cells of female α4 knockout (KO) mice were tested for plasmalemmal levels of the δ subunit within dendritic spine and shaft profiles at the onset of puberty. EM-ICC revealed that the α4 and δ subunits localize on dendritic spines and shafts at sites extrasynaptic to GABAergic input at pubertal onset in tissue of wild-type (WT) mice. At pubertal onset, plasmalemmal localization of the δ subunit is reduced 45.9% on dendritic spines, and 56.7% on dendritic shafts with KO of the α4 subunit, as compared to WT tissue, yet levels of intracellular δ immunoreactivity remain unchanged. The decline in plasmalemmal localization is manifested as decreased responsiveness to the GABA agonist gaboxadol at concentrations that are selective for δ-containing GABARs. Additionally, α4 KO mice have larger dendritic spine and shaft profiles. Our findings demonstrate that α4 subunit expression strongly influences the pubertal increase of δ subunits at the plasma membrane, and that genetic deletion of α4 serves as a functional knock-down of δ-containing GABARs.

GABA acts as a ligand chaperone in the early secretory pathway to promote cell surface expression of GABAA receptors.

GABA (gamma-aminobutyric acid) is the primary inhibitory neurotransmitter in brain. The fast inhibitory effect of GABA is mediated through the GABA(A) receptor, a postsynaptic ligand-gated chloride channel. We propose that GABA can act as a ligand chaperone in the early secretory pathway to facilitate GABA(A) receptor cell surface expression. Forty-two hours of GABA treatment increased the surface expression of recombinant receptors expressed in HEK 293 cells, an effect accompanied by an increase in GABA-gated chloride currents. In time-course experiments, a 1h GABA exposure, followed by a 5h incubation in GABA-free medium, was sufficient to increase receptor surface expression. A shorter GABA exposure could be used in HEK 293 cells stably transfected with the GABA transporter GAT-1. In rGAT-1HEK 293 cells, the GABA effect was blocked by the GAT-1 inhibitor NO-711, indicating that GABA was acting intracellularly. The effect of GABA was prevented by brefeldin A (BFA), an inhibitor of early secretory pathway trafficking. Coexpression of GABA(A) receptors with the GABA synthetic enzyme glutamic acid decarboxylase 67 (GAD67) also resulted in an increase in receptor surface levels. GABA treatment failed to promote the surface expression of GABA binding site mutant receptors, which themselves were poorly expressed at the surface. Consistent with an intracellular action of GABA, we show that GABA does not act by stabilizing surface receptors. Furthermore, GABA treatment rescued the surface expression of a receptor construct that was retained within the secretory pathway. Lastly, the lipophilic competitive antagonist (+)bicuculline promoted receptor surface expression, including the rescue of a secretory pathway-retained receptor. Our results indicate that a neurotransmitter can act as a ligand chaperone in the early secretory pathway to regulate the surface expression of its receptor. This effect appears to rely on binding site occupancy, rather than agonist-induced structural changes, since chaperoning is observed with both an agonist and a competitive antagonist.

A critical role for alpha4betadelta GABAA receptors in shaping learning deficits at puberty in mice.

The onset of puberty defines a developmental stage when some learning processes are diminished, but the mechanism for this deficit remains unknown. We found that, at puberty, expression of inhibitory alpha4betadelta gamma-aminobutyric acid type A (GABAA) receptors (GABAR) increases perisynaptic to excitatory synapses in CA1 hippocampus. Shunting inhibition via these receptors reduced N-methyl-D-aspartate receptor activation, impairing induction of long-term potentiation (LTP). Pubertal mice also failed to learn a hippocampal, LTP-dependent spatial task that was easily acquired by delta-/- mice. However, the stress steroid THP (3alphaOH-5alpha[beta]-pregnan-20-one), which reduces tonic inhibition at puberty, facilitated learning. Thus, the emergence of alpha4betadelta GABARs at puberty impairs learning, an effect that can be reversed by a stress steroid.

Plasticity of the alpha4betadelta GABAA receptor.

The GABAR [GABA(A) (gamma-aminobutyric acid type A) receptor], which mediates most inhibition in the brain, is regulated homoeostatically to maintain an optimal level of neuronal excitability. In particular, the alpha(4)betadelta subtype of the GABAR plays a pivotal role in this regulation. This receptor, which is expressed extrasynaptically on the dendrites, normally has low expression in the brain, but displays a remarkable degree of plasticity. It can also be a sensitive target for endogenous neurosteroids such as THP (3alpha-hydroxy-5[alpha]beta-pregnan-20-one (allo-pregnanolone); a neurosteroid and positive modulator of the GABAR), which is released during stress, although the effect of the steroid is polarity-dependent, such that it increases inward current, but decreases outward current, at alpha(4)beta(2)delta GABAR. Expression of alpha(4)beta(2)delta GABAR in CA1 hippocampus is also tightly regulated by fluctuating levels of neurosteroids, as seen at the onset of puberty. Declining levels of inhibition resulting from the decrease in THP at puberty are compensated for by an increase in alpha(4)betadelta GABAR along the apical dendrites of CA1 hippocampal pyramidal cells, which reduces neuronal excitability by decreasing the input resistance. However, excessive decrease of neuronal function is averted when THP levels rise, as would occur during stress, because this steroid decreases the outward GABAergic tonic current via inhibition of alpha(4)beta(2)delta GABAR, thereby restoring measures of neuronal excitability to pre-pubertal levels. Thus the homoeostatic regulation of alpha(4)betadelta GABAR expression plays an important role in maintaining ambient levels of neuronal excitability at puberty.

Puberty, steroids and GABA(A) receptor plasticity.

GABA(A) receptors (GABAR) mediate most inhibition in the CNS and are also a target for neuroactive steroids such as 3alpha,5[alpha]beta-THP (3alphaOH-5[alpha]beta-OH-pregnan-20-one or [allo]pregnanolone). Although these steroids robustly enhance current gated by alpha1beta2delta GABAR, we have shown that 3alpha,5[alpha]beta-THP effects at recombinant alpha4beta2delta GABAR depend on the direction of Cl(-) flux, where the steroid increases outward flux, but decreases inward flux through the receptor. This polarity-dependent inhibition of alpha4beta2delta GABAR resulted from an increase in the rate and extent of rapid desensitization of the receptor, recorded from recombinant receptors expressed in HEK-293 cells with whole cell voltage clamp techniques. This inhibitory effect of 3alpha,5[alpha]beta-THP was not observed at other receptor subtypes, suggesting it was selective for alpha4beta2delta GABAR. Furthermore, it was prevented by a selective mutation of basic residue arginine 353 in the intracellular loop of the receptor, suggesting that this might be a putative chloride modulatory site. Expression of alpha4betadelta GABAR increases markedly at extrasynaptic sites at the onset of puberty in female mice. At this time, 3alpha,5[alpha]beta-THP decreased the inhibitory tonic current, recorded with perforated patch techniques to maintain the physiological Cl(-) gradient. By decreasing this shunting inhibition, 3alpha,5[alpha]beta-THP increased the excitability of CA1 hippocampal pyramidal cells at puberty. These effects of the steroid were opposite to those observed before puberty when 3alpha,5[alpha]beta-THP reduced neuronal excitability as a pre-synaptic effect. Behaviorally, the excitatory effect of 3alpha,5[alpha]beta-THP was reflected as an increase in anxiety at the onset of puberty in female mice. Taken together, these findings suggest that the emergence of alpha4beta2delta GABAR at the onset of puberty reverses the effect of a stress steroid. These findings may be relevant for the mood swings and increased response to stressful events reported in adolescence.