Memories are formed at structures in the brain known as dendritic spines. These structures receive connections from other brain cells through regions called synapses. In humans, the number of these brain connections increases dramatically from birth to childhood, reflecting a period of rapid learning. However, the number of these brain connections halves after puberty, a dramatic reduction shown in many brain areas and for many species, including humans and rodents. This process is referred to as adolescent synaptic pruning and is thought to be important for optimal learning in adulthood because it is disrupted in autism and schizophrenia. Synaptic pruning is believed to remove unnecessary brain connections to make room for new relevant memories. However, the process that triggers synaptic pruning is not known.

Within the brain, proteins called inhibitory GABA receptors are targets for chemicals that reduce the activity of nerve cells. As brain connections must be kept active to survive, inhibitory receptors could help to trigger synaptic pruning.

Afroz, Parato et al. now show that, at puberty, the number of a particular type of GABA_A receptor increases in the brain of female mice. This triggers synaptic pruning in the hippocampus, a key brain area necessary for learning and memory. By reducing brain activity, these inhibitory receptors also reduce the levels of a protein in the dendritic spine that stabilizes the scaffolding of the spine to maintain its structure.

Mice that do not have these GABA_A receptors maintain a constant high level of brain connections throughout adolescence, and synaptic pruning does not occur in their brains. These mice were initially able to learn to avoid a specific location that provided a mild shock to their foot. However, when this location changed the mice were unable to re-learn where to avoid, suggesting that too many brain connections limits learning potential.

Brain connections are regulated by many factors, including the environment and stress. Future studies will test how these additional factors alter synaptic pruning in adolescence, and will test drugs that target these inhibitory receptors to manipulate adolescent pruning. These findings may suggest new treatments for “normalizing” synaptic pruning in conditions where this process occurs abnormally, such as autism and schizophrenia.

During the pubertal period, the density of dendritic spines decreases by half in widespread areas of the CNS (Huttenlocher, 1979; Zehr et al., 2006; Petanjek et al., 2011; Koss et al., 2014), including the CA1 hippocampus and temporal lobe of both rodents (Yildirim et al., 2008) and humans (Tang et al., 2014), sites essential for learning and memory (Pastalkova, 2006). Dendritic spines express NMDA receptors (NMDARs) at excitatory synapses (Matsuzaki et al., 2004) which can be activated to form memory traces (Bannerman et al., 2008). A modelling study (Ruppin, 1999) suggests that an optimal spine density, produced by developmental pruning of unnecessary synapses, may be necessary not only for the ability to form memories but also the ability to re-learn or 'update' previously learned information. Despite the implied importance of synaptic pruning during adolescence, the mechanisms underlying spine elimination in CA1 hippocampus during puberty are not yet known nor are the behavioral outcomes of altered spine density.

At certain times in development, scavenging by immune system components such as the microglia plays a role in spine pruning (Schafer et al., 2012; Sekar et al., 2016). This system is likely the final step in synapse elimination in several CNS areas including the CA3 hippocampus, but does not have a role in synapse loss of CA1 hippocampal pyramidal cells during puberty (Shi et al., 2015).

One factor not yet considered in adolescent pruning is the role of inhibition in the brain mediated by GABA_A receptors (GABARs). GABARs mediate most inhibition in the brain and are pentameric membrane proteins, of diverse sub-type, which conduct a Cl^- current. In the hippocampus, GABARs are either expressed sub-synaptically, where they generate a phasic current, or extrasynaptically, where they underlie a tonic inhibitory conductance (Stell and Mody, 2002).

During the pubertal period (PND 35–44) of female mice, we have shown that α4βδ GABARs transiently emerge on dendritic spines of CA1 pyramidal cells, adjacent to excitatory synapses (Shen et al., 2010; Aoki et al., 2012). These extrasynaptic receptors, which are sensitive to low levels of ambient GABA (<1 μM) (Brown et al., 2002), generate a shunting inhibition which reduces NMDAR-generated current (Shen et al., 2010). However, NMDA current is robust in pubertal mice lacking expression of α4βδ GABARs (Shen et al., 2010), suggesting that the inhibition mediated by α4βδ GABARs produces the impairment rather than a lack of functional NMDARs at puberty.

We have also shown that the pubertal increase in hippocampal α4βδ GABAR expression prevents induction of long-term potentiation, an in vitro model of learning, and impairs spatial learning of female mice (Shen et al., 2010; Shen et al., 2016). These deficits were not observed at puberty in α4 KO (Shen et al., 2016) or δ KO (Shen et al., 2010) mice, implicating pubertal α4βδ GABARs as the mediating factor.

We have extended these studies to show here that expression of α4βδ GABARs at the onset of puberty initiates synaptic pruning in the female mouse hippocampus, which ultimately reduces spine density post-pubertally (comparing spine density at PND 35 versus PND 56). In the α4 KO mouse, pruning does not take place and the cognitive ability of the post-pubertal mice is impaired. We also show α4βδ and NMDAR involvement in the pruning process by the administration of selective drugs during the pubertal period (PND 35–44) to determine effects on spine density post-pubertally (PND 56). We further suggest that α4βδ-triggered pruning is due to impairment of NMDAR activation which regulates expression of kalirin-7 (Kal7), a Rho guanine nucleotide exchange factor (GEF) important for stabilizing the actin cytoskeleton (Penzes et al., 2001).

Pubertal synaptic pruning is seen in many CNS areas (Huttenlocher, 1979; Zehr et al., 2006; Petanjek et al., 2011), and is correlated with EEG changes in humans (Campbell et al., 2012), where the steepest reduction in slow wave (delta, 1–4 Hz) activity occurs at the onset of puberty. Our findings suggest that α4βδ GABARs play a critical role in this process. α4βδ GABARs are uniquely localized to spines during puberty where they reduce NMDAR current (Shen et al., 2010), necessary for spine stability (Alvarez et al., 2007). In contrast, blockade of the predominant extrasynaptic GABAR in CA1 hippocampus, α5β3γ2, did not facilitate NMDAR activation or alter synaptic pruning during the pubertal period, nor did modulation of synaptic γ2-containing GABARs with 200 nM SR95531 or lorazepam. Thus, these findings suggest that α4βδ GABARs selectively trigger synaptic pruning at puberty via impairment of NMDAR activation. This is in contrast to visual cortex, where synaptic α1β2γ2 GABARs target dendritic spines (Kawaguchi and Kubota, 1997), and play a role in spine maturation (Heinen et al., 2003). Pubertal synaptic pruning is not dependent upon the ovarian hormone estradiol (Yildirim et al., 2008), which in fact increases spine density (Woolley et al., 1997).

In the present study, MK-801 administration during the pubertal period of female mice increased the density of dendritic spines post-pubertally, resulting in increases in both mushroom and stubby spine-types. A recent study (Han et al., 2013) reports that similar MK-801 treatment across the peri-pubertal period in male rats also increases the density of stubby spines in hippocampus, but decreases the mushroom spine-types. The reason for the disparity in the two outcomes may be due to gender or developmental differences, as the latter study used rats across an age range that likely includes both pre-pubertal and pubertal ages (Marty et al., 2001).

Although MK-801 is an NMDA antagonist, it has been shown to increase NMDAR expression, as a compensatory effect 24 hr after administration (0.1–1.0 mg/kg), selectively in the CA1 region of the hippocampus (Gao and Tamminga, 1995). This is the most likely mechanism by which MK-801 increased spine density in the present study where injections were administered once a day during the pubertal period. In the pre-frontal cortex, only a single low dose of MK-801 increases NMDAR expression on pyramidal cells (Xi et al., 2009), while higher doses have no effect, but decrease NMDAR expression and AMPA receptor-mediated currents of fast-spiking interneurons (Wang and Gao, 2012) suggesting that effects of this drug are cell-type and region specific. Other studies have suggested that peri-adolescent administration of MK-801 alters GABAergic circuits in adulthood (Thomases et al., 2013). We cannot rule out the additional possibility that the MK-801 treatment also produced changes in the GABAergic circuitry of the hippocampus which may have contributed to the observed changes in spine density post-pubertally.

α4βδ GABAR-induced impairment of NMDAR activation at puberty reduces expression of Kal7, a spine protein involved in spine restructuring which binds to the post-synaptic density (PSD) (Penzes et al., 2001). Kal7 activates the small GTPase Rac1 which regulates the actin cytoskeleton via P21-activated kinases within the spine (Ma et al., 2014). Although many proteins localize to the spine, Kal7 is one of the few shown to be necessary for maintenance of existing spines (Penzes et al., 2001; Ma et al., 2003). That may explain results from the present study where a 50% reduction in Kal7 expression at puberty resulted in a 50% decrease in spine number. The fact that synaptic pruning was prevented in the absence of Kal7 expression suggests that dynamic regulation of Kal7 expression during adolescence may be one factor underlying synaptic pruning. However, many other spine proteins have been identified which alter spine density, including shank3, IQGAP1, valosin, axin and NEDD9 (Roussignol et al., 2005; Gao et al., 2011; Wiesmann et al., 1975; Knutson et al., 2016; Chen et al., 2015). Thus, synaptic remodeling during adolescence may incorporate a more complex array of spine protein changes.

Several studies have suggested that scavenging by microglia (Schafer et al., 2012) or direct autophagy (Tang et al., 2014) prunes spines. In particular, the C4 complement system is abnormal in schizophrenia; C4 activates C3, which binds to target spines and promotes their engulfment by phagocytic cells (Sekar et al., 2016). This system may be target and developmentally specific – as it does not underlie adolescent pruning in CA1 hippocampus (Shi et al., 2015). In addition, the involvement of the complement cascade in synaptic pruning would likely be the final step in spine removal and does not preclude involvement of other systems, such as NMDAR inhibition by α4βδ receptors which would be the initial trigger for the pruning process.

The mushroom spines were selectively removed during pubertal synaptic pruning in the wild-type mouse, leaving an abundance of thin spines. In order to better understand the role of the various spine types in cognition, we directly examined the changes in spine morphology produced by learning and re-learning a spatial task in the wild-type mouse. Our results suggest that learning increased mushroom spines, consistent with earlier reports (Beltrán-Campos et al., 2011; González-Ramírez et al., 2014), which we also observed by 2 hr after re-learning, when the density of thin spines also decreased. These two spine types are well-characterized and sub-serve different functions: Thin spines express abundant numbers of NMDARs, are highly motile and plastic (Kasai et al., 2003; Holtmaat et al., 2006), while the larger mushroom spines are relatively more stable and express AMPA receptors predominantly (Dumitriu et al., 2010; Matsuzaki et al., 2001). Recent studies using two photon technology to assess changes in spines produced by NMDAR-dependent LTP, an in vitro model of learning, have shown that smaller 'thin' spines develop enlarged heads and resemble 'mushroom' spines following LTP induction, accompanied by increased expression of AMPA receptors (Hill and Zito, 2013; Kopec et al., 2006; Harvey and Svoboda, 2007). These larger spine heads have larger post-synaptic densities and active zones, consistent with stronger synaptic connections (Bell et al., 2014). Typically, these changes are first observed within 15 min. with peak effects 40 min to 2 hr after LTP induction, similar to the time-course of our effects.

Our findings suggest that a high spine density produces cognitive impairment. Although modelling studies have predicted the cognitive outcome of increases in spine density (Ruppin, 1999), most experimental studies to date have only examined the effect of spine loss on cognition (Oaks et al., 2016). The high spine density in the unpruned condition in the present study selectively impaired re-learning, while learning a spatial task was similar to controls. The impairments in re-learning in the unpruned conditions may be due to the higher prevalence of mushroom-type spines and lower density of thin spine-types. Mushroom spine density was only increased above its already elevated level in the unpruned mouse following the learning trial. No additional increases in mushroom spines were observed for the unsuccessful re-learning trial. There may be a maximum number of mushroom spines that can be supported by a dendritic segment due to the energy requirement necessary for spine maintenance or due to spatial constraints. Alternatively, the lower density of thin spines may have been insufficient to permit successful re-learning.

The changes in spine density observed after learning and re-learning protocols were of the same magnitude as those observed during adolescence, reflecting the high plasticity of spines, although changes occurred in the opposite direction. Changes in spine types were distinct for the two events as well: during learning and re-learning, mushroom spine-types increased, while these spine-types decreased during adolescence. In addition, the spine density of the control, post-pubertal mice may reflect the lower limit of synapse number because these mice were housed in non-enriched cages with limited sensory stimuli. Numerous studies have shown that exposure to more complex environments, even for 1 hr per day, increases spine density in many areas of the CNS, including CA1 hipppocampus (Turner and Greenough, 1985; Jung and Herms, 2014; Kitanishi et al., 2009). Recent studies report that environmental enrichment increases large spines and enhances spine turnover (Jung and Herms, 2014; Kitanishi et al., 2009), suggesting that spine dynamics are environmentally specific.

LTD induction was also impaired in the unpruned condition (α4 KO), consistent with other reports which describe impairment in LTD under conditions where thin spine number is reduced (Spiga et al., 2014). Two photon studies have indeed shown that this protocol reduces thin spine number (Nägerl et al., 2004), thus suggesting that a critical number of thin spines may be necessary for LTD induction. LTD has been suggested as a cellular mechanism underlying synaptic changes necessary for re-learning (Nicholls et al., 2008), where weaker synaptic connections mediated by thin spines are reduced. In contrast to the present results, studies reporting more massive decreases in thin spine number, as a result of aging or chronic alcohol exposure, also observed impairments in learning and LTP (Dumitriu et al., 2010; Spiga et al., 2014), suggesting that the critical number of thin spines necessary for learning is below that required for relearning.

The results from the present study may have relevance for the cognitive impairment in autism and schizophrenia where synaptic pruning is abnormal (Hutsler and Zhang, 2010; van Spronsen and Hoogenraad, 2010). Adolescent synaptic pruning of the temporal lobe does not occur in autism (Tang et al., 2014), leaving an abundance of dendritic spines (Hutsler and Zhang, 2010) which are associated with impairments in reversal learning (D'Cruz et al., 2013), similar to our results in the α4 KO mouse. In fact, reduced α4 expression has been reported in autism (Fatemi et al., 2010), which is correlated with increased risk of developing this disorder (Collins et al., 2006), although identified genetic abnormalities in α4 and/or δ genes in autism and schizophrenia are relatively rare (Ma et al., 2005; Bullock et al., 2008). Both disorders are more likely to occur in males (Croen et al., 2002; Iacono and Beiser, 1992), for which we also show α4βδ involvement in adolescent pruning. Initial deficits of autism appear in early childhood, but loss of cognitive gains are frequently reported in adolescence following improvement earlier in development (Sigman and McGovern, 2005; Gillberg and Steffenburg, 1987). Thus, the lack of synaptic pruning in adolescence may contribute to this developmental slow-down. The results from the present study may suggest novel therapeutic strategies to normalize disrupted synaptic pruning in these disorders.

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Author details

1. Sonia Afroz

   1. Department of Physiology and Pharmacology, SUNY Downstate Medical Center, Brooklyn, United States
   2. Program in Neural and Behavioral Science, SUNY Downstate Medical Center, Brooklyn, United States

   Present address

   Department of Biomedical Sciences, University of California, Riverside, United States

   Contribution

   SA, Performed the spine density/morphological analyses and behavioral experiments, Analyzed the data and acquired images for +/+ and α4-/- and drug-treated mice, Contributed to the experimental design, Conception and design, Acquisition of data, Analysis and interpretation of data

   Contributed equally with

   Julie Parato

   Competing interests

   The authors declare that no competing interests exist.

2. Julie Parato

   1. Department of Physiology and Pharmacology, SUNY Downstate Medical Center, Brooklyn, United States
   2. Program in Neural and Behavioral Science, SUNY Downstate Medical Center, Brooklyn, United States

   Contribution

   JP, Performed the spine density/morphological analyses and behavioral experiments for the males and some of the drug groups, Performed the immunohistochemical experiments, Contributed to the experimental design, Conception and design, Acquisition of data, Analysis and interpretation of data

   Contributed equally with

   Sonia Afroz

   Competing interests

   The authors declare that no competing interests exist.

3. Hui Shen

   1. Department of Physiology and Pharmacology, SUNY Downstate Medical Center, Brooklyn, United States
   2. School of Biomedical Engineering, Tianjin Medical University, Tianjin, China

   Contribution

   HS, Performed the electrophysiological experiments, Acquisition of data

   Competing interests

   The authors declare that no competing interests exist.

4. Sheryl Sue Smith

   1. Department of Physiology and Pharmacology, SUNY Downstate Medical Center, Brooklyn, United States
   2. The Robert F. Furchgott Center for Neural and Behavioral Science, SUNY Downstate Medical Center, Brooklyn, United States

   Contribution

   SSS, Designed the experiments, Constructed the figures, Analysis and interpretation of data, Wrote the paper

   For correspondence

   sheryl.smith@downstate.edu

   Competing interests

   The authors declare that no competing interests exist.

   "This ORCID iD identifies the author of this article:" 0000-0003-4308-3267

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