Patrizia Panzanelli

Synaptic pruning by microglia is necessary for normal brain development.

A good brain needs a good vacuum cleaner. Microglia are highly motile phagocytic cells that infiltrate and take up residence in the developing brain, where they are thought to provide a surveillance and scavenging function. However, although microglia have been shown to engulf and clear damaged cellular debris after brain insult, it remains less clear what role microglia play in the uninjured brain. Here, we show that microglia actively engulf synaptic material and play a major role in synaptic pruning during postnatal development in mice. These findings link microglia surveillance to synaptic maturation and suggest that deficits in microglia function may contribute to synaptic abnormalities seen in some neurodevelopmental disorders.

The γ2 Subunit of GABAA Receptors Is a Substrate for Palmitoylation by GODZ

The neurotransmitter GABA activates heteropentameric GABAA receptors, which are composed mostly of α, β, and γ2 subunits. Regulated membrane trafficking and subcellular targeting of GABAA receptors is important for determining the efficacy of GABAergic inhibitory function. Of special interest is the γ2 subunit, which is mostly dispensable for assembly and membrane insertion of functional receptors but essential for accumulation of GABAA receptors at synapses. In a search for novel receptor trafficking proteins, we have used the SOS-recruitment system and isolated a Golgi-specific DHHC zinc finger protein (GODZ) as a novel γ2 subunit-interacting protein. GODZ is a member of the superfamily of DHHC cysteine-rich domain (DHHC-CRD) polytopic membrane proteins shown recently in yeast to represent palmitoyltransferases. GODZ mRNA is found in many tissues; however, in brain the protein is detected in neurons only and highly concentrated and asymmetrically distributed in the Golgi complex. GODZ interacts with a cysteine-rich 14-amino acid domain conserved specifically in the large cytoplasmic loop of γ1-3 subunits but not in other GABAA receptor subunits. Coexpression of GODZ and GABAA receptors in heterologous cells results in palmitoylation of the γ2 subunit in a cytoplasmic loop domain-dependent manner. Neuronal GABAA receptors are similarly palmitoylated. Thus, GODZ-mediated palmitoylation represents a novel posttranslational modification that is selective forγ subunit-containing GABAA receptor subtypes, a mechanism that is likely to be important for regulated trafficking of these receptors in the secretory pathway.

Colocalization of multiple GABAA receptor subtypes with gephyrin at postsynaptic sites

Clustering of gamma aminobutyric acid (GABA)A receptors to postsynaptic sites requires the presence of both the γ2 subunit and gephyrin. Here, we analyzed by double‐immunofluorescence staining the colocalization of gephyrin and major GABAA‐receptor subtypes distinguished by the subunits α1, α2, α3, or γ2 in adult rat brain. By using confocal laser scanning microscopy, GABAA‐receptor subunit staining revealed brightly stained clusters that were colocalized with gephyrin‐positive clusters of similar size and distribution in several brain regions, including cerebellum, hippocampus, thalamus, and olfactory bulb. In addition, a diffuse staining was observed for GABAA‐receptor subunits in the neuropil, presumably representing extrasynaptic receptors. Overall, only few gephyrin‐positive clusters were not colocalized with GABAA‐receptor subunit clusters. Electron microscopic analysis in cerebellar cortex confirmed the selective postsynaptic localization of gephyrin. High‐resolution images (voxel size, 50 × 50 × 150 nm) were restored with an iterative image deconvolution procedure based on a measured point‐spread function to analyze the colocalization between GABAA‐receptor subunits and gephyrin in individual clusters. This analysis revealed a considerable heterogeneity in the micro‐organization of these presumptive GABAergic postsynaptic sites. For instance, whereas gephyrin‐ and γ2 subunit‐positive clusters largely overlapped in the cerebellar molecular layer, the colocalization was only partial in glomeruli of the granule cell layer, where small gephyrin clusters typically were “embedded” in larger GABAA‐receptor clusters. These findings show that gephyrin is associated with a majority of GABAA‐receptor subtypes in brain, and document the usefulness of image deconvolution for analyzing the structural organization of the postsynaptic apparatus by fluorescence microscopy. J. Comp. Neurol. 420:481–498, 2000.

Profilin2 contributes to synaptic vesicle exocytosis, neuronal excitability, and novelty‐seeking behavior

Profilins are actin binding proteins essential for regulating cytoskeletal dynamics, however, their function in the mammalian nervous system is unknown. Here, we provide evidence that in mouse brain profilin1 and profilin2 have distinct roles in regulating synaptic actin polymerization with profilin2 preferring a WAVE‐complex‐mediated pathway. Mice lacking profilin2 show a block in synaptic actin polymerization in response to depolarization, which is accompanied by increased synaptic excitability of glutamatergic neurons due to higher vesicle exocytosis. These alterations in neurotransmitter release correlate with a hyperactivation of the striatum and enhanced novelty‐seeking behavior in profilin2 mutant mice. Our results highlight a novel, profilin2‐dependent pathway, regulating synaptic physiology, neuronal excitability, and complex behavior.

GABAA receptors and plasticity of inhibitory neurotransmission in the central nervous system.

GABAA receptors (GABAARs) are ligand‐gated Cl− channels that mediate most of the fast inhibitory neurotransmission in the central nervous system (CNS). Multiple GABAAR subtypes are assembled from a family of 19 subunit genes, raising the question of the significance of this heterogeneity. In this review, we discuss the evidence that GABAAR subtypes represent distinct receptor populations with a specific spatio‐temporal expression pattern in the developing and adult CNS, being endowed with unique functional and pharmacological properties, as well as being differentially regulated at the transcriptional, post‐transcriptional and translational levels. GABAAR subtypes are targeted to specific subcellular domains to mediate either synaptic or extrasynaptic transmission, and their action is dynamically regulated by a vast array of molecular mechanisms to adjust the strength of inhibition to the changing needs of neuronal networks. These adaptations involve not only changing the gating or kinetic properties of GABAARs, but also modifying the postsynaptic scaffold organised by gephyrin to anchor specific receptor subtypes at postsynaptic sites. The significance of GABAAR heterogeneity is particularly evident during CNS development and adult neurogenesis, with different receptor subtypes fulfilling distinct steps of neuronal differentiation and maturation. Finally, analysis of the specific roles of GABAAR subtypes reveals their involvement in the pathophysiology of major CNS disorders, and opens novel perspectives for therapeutic intervention. In conclusion, GABAAR subtypes represent the substrate of a multifaceted inhibitory neurotransmission system that is dynamically regulated and performs multiple operations, contributing globally to the proper development, function and plasticity of the CNS.

Immunofluorescence in brain sections: simultaneous detection of presynaptic and postsynaptic proteins in identified neurons

Elucidating the molecular organization of synapses is essential for understanding brain function and plasticity. Immunofluorescence, combined with various fluorescent probes, is a sensitive and versatile method for morphological studies. However, analysis of synaptic proteins in situ is limited by epitope-masking after tissue fixation. Furthermore, postsynaptic proteins (such as ionotropic receptors and scaffolding proteins) often require weaker fixation for optimal detection than most intracellular markers, thereby hindering simultaneous visualization of these molecules. We present three protocols, which are alternatives to perfusion fixation, to overcome these restrictions. Brief tissue fixation shortly after interruption of vital functions preserves morphology and antigenicity. Combined with specific neuronal markers, selective detection of γ-aminobutyric acid A (GABAA) receptors and the scaffolding protein gephyrin in relation to identified inhibitory presynaptic terminals in the rodent brain is feasible by confocal laser scanning microscopy. The most sophisticated of these protocols can be associated with electrophysiology for correlative studies of synapse structure and function. These protocols require 2–3 consecutive days for completion.

Distinct mechanisms regulate GABAA receptor and gephyrin clustering at perisomatic and axo-axonic synapses on CA1 pyramidal cells

Non‐Technical Summary  To be effective, synaptic transmission requires precise alignment of the presynaptic terminal, releasing the neurotransmitter, with the postsynaptic density, where receptors are present at high density. Complex molecular mechanisms ensure this interplay between neurons and, in addition, stabilize receptors in the postsynaptic membrane. To explore these mechanisms at GABAergic synapses, which mediate inhibitory neurotransmission in the brain, we investigated here the consequences of ‘removing’ the receptors, using targeted gene deletion. Our results show that the receptors are dispensable for synapse formation, but are required for the postsynaptic aggregation of several proteins involved in receptor trafficking, anchoring and regulation. Defects in the molecular regulation of GABAergic synapses have been associated with neurodevelopmental disorders, mental retardation, anxiety and mood disorders, underscoring the relevance of fine tuning of GABAergic inhibition for proper brain function.

Presynaptic co-localization of carnosine and glutamate in olfactory neurones.

OLFACTION plays a dominant role in modulating behaviour in most vertebrate species and the olfactory bulb is considered a model system for characterizing principles of neural computation. Nevertheless, although the physiology and neurochemistry of the olfactory circuits have been widely studied, the neurotransmitter released by olfactory receptor neurones remains unknown. We now describe the ultrastructural localization of the dipeptide carnosine and the excitatory amino acid glutamate in the glomerular layer of the mouse olfactory bulb. We demonstrate that both carnosine-like and glutamate-like immunoreactivities are selectively co-localized in the olfactory neurone boutons. These observations, taken with the recent findings of glutamate-receptor subunit expression in rodent olfactory bulb, argue compellingly for a role of glutamate in olfactory neurotransmission and suggest a modulatory effect of carnosine.

GABAergic inhibition at dendrodendritic synapses tunes gamma oscillations in the olfactory bulb.

In the olfactory bulb (OB), odorants induce oscillations in the γ range (20–80 Hz) that play an important role in the processing of sensory information. Synaptic transmission between dendrites is a major contributor to this processing. Glutamate released from mitral cell dendrites excites the dendrites of granule cells, which in turn mediate GABAergic inhibition back onto mitral cells. Although this reciprocal synapse is thought to be a key element supporting oscillatory activity, the mechanisms by which dendrodendritic inhibition induces and maintains γ oscillations remain unknown. Here, we assessed the role of the dendrodendritic inhibition, using mice lacking the GABAA receptor α1-subunit, which is specifically expressed in mitral cells but not in granule cells. The spontaneous inhibitory postsynaptic current frequency in these mutants was low and was consistent with the reduction of GABAA receptor clusters detected by immunohistochemistry. The remaining GABAA receptors in mitral cells contained the α3-subunit and supported slower decaying currents of unchanged amplitude. Overall, inhibitory-mediated interactions between mitral cells were smaller and slower in mutant than in WT mice, although the strength of sensory afferent inputs remained unchanged. Consequently, both experimental and theoretical approaches revealed slower γ oscillations in the OB network of mutant mice. We conclude, therefore, that fast oscillations in the OB circuit are strongly constrained by the precise location, subunit composition and kinetics of GABAA receptors expressed in mitral cells.

The actin‐binding protein profilin I is localized at synaptic sites in an activity‐regulated manner

Morphological changes at synaptic specializations have been implicated in regulating synaptic strength. Actin turnover at dendritic spines is regulated by neuronal activity and contributes to spine size, shape and motility. The reorganization of actin filaments requires profilins, which stimulate actin polymerization. Neurons express two independent gene products − profilin I and profilin II. A role for profilin II in activity‐dependent mechanisms at spine synapses has recently been described. Although profilin I interacts with synaptic proteins, little is known about its cellular and subcellular localization in neurons. Here, we investigated the subcellular distribution of this protein in brain neurons as well as in hippocampal cultures. Our results indicate that the expression of profilin I varies in different brain regions. Thus, in cerebral cortex and hippocampus profilin I immunostaining was associated predominantly with dendrites and was present in a subset of dendritic spines. In contrast, profilin I in cerebellum was associated primarily with presynaptic structures. Profilin I immunoreactivity was partially colocalized with the synaptic molecules synaptophysin, PSD‐95 and gephyrin in cultured hippocampal neurons, indicating that profilin I is present in only a subset of synapses. At dendritic spine structures, profilin I was found primarily in protrusions, which were in apposition to presynaptic terminal boutons. Remarkably, depolarization with KCl caused a moderate but significant increase in the number of synapses containing profilin I. These results show that profilin I can be present at both pre‐ and postsynaptic sites and suggest a role for this actin‐binding protein in activity‐dependent remodelling of synaptic structure.