Functional Synaptogenesis Research Articles

Chemical Treatments for Insect Cell Differentiation: The Effects of 20-Hydroxyecdysone and Veratridine on Cultured Spodoptera frugiperda (Sf21) Insect Cell Ultrastructure

Simple SummaryCultured insect Sf21 cells treated with the hormone 20-hydroxyecdysone grow long processes and resemble neurons. They also make physical contact with one another and appear to have the potential to form synapses, areas in which nerve cells are in close contact and communicate with one another electrically or by the release of chemical transmitters. This study uses electron microscopy to look for structural evidence of synapses in 20-hydroxyexdysone treated Sf21 cell cultures. Unfortunately, no evidence of synaptic structures were observed, suggesting that other factors are required for the formation of functional synapses in these cultures.Previous studies have shown that insect cell cultures stop dividing, form clumps, and can be induced to grow processes reminiscent of axons, when the culture medium is supplemented with 20-hydroxyecdysone, insulin, or an agent that mimics their action, such as the ecdysone agonist, methoxyfenozide. Those cell growing processes resemble nerve cells, and the present study evaluates the ultrastructure of these cultures by transmission electron microscopy. Sf21 cells treated with 20-hydroxyecdysone (with or without veratridine amendment) and subjected to ultrastructural analysis had a similar somatic appearance to control cells, with slight changes in organelles and organization, such as a greater number of cytoplasmic vacuoles and mitochondrial granules. Finger-like projections were observed between control and treated cells. However, no structural markers of synaptic contacts (e.g., vesicles or synaptic thickenings) were observed in controls, 20-hydroxyecdysone, or 20-hydroxyecdysone + veratridine treated cells. It is concluded that additional agents would be required to induce functional synaptogenesis in Sf21 cells.

Developmental roles of the spontaneous depolarization wave in synaptic network formation in the embryonic brainstem

One of the earliest activities expressed within the developing central nervous system is a widely propagating wave-like activity, which we referred to as the depolarization wave. Despite considerable consensus concerning the global features of the activity, its physiological role is yet to be clarified. The depolarization wave is expressed during a specific period of functional synaptogenesis, and this developmental profile has led to the hypothesis that the wave plays some roles in synaptic network organization. In the present study, we tested this hypothesis by inhibiting the depolarization wave in ovo and examining its effects on the development of functional synapses in vagus nerve-related brainstem nuclei of the chick embryo. Chronic inhibition of the depolarization wave had no significant effect on the developmental time course, amplitude, and spatial distribution of monosynaptic excitatory postsynaptic potentials in the first-order nuclei of the vagal sensory pathway (the nucleus of the tractus solitarius (NTS) and the contralateral non-NTS region), but reduced polysynaptic responses in the higher-order nucleus (the parabrachial nucleus). These results suggest that the depolarization wave plays an important role in the initial process of functional synaptic expression in the brainstem, especially in the higher-order nucleus of the cranial sensory pathway.

Structural divergence of essential triad ribbon synapse proteins among placental mammals – Implications for preclinical trials in photoreceptor transplantation therapy

As photoreceptor transplantation rapidly moves closer to the clinic, verifying graft efficacy in animal models may have unforeseen xenogeneic barriers. Although photoreceptor transplants have most convincingly exhibited functional synaptogenesis in conspecific studies, such evidence (while ruling out false-positives due to: viral graft labeling, fusion/cytosolic transfer, or neuroprotection) has not yet been shown for discordant xenografts. From this, a fundamental question should be raised: is useful xenosynaptogenesis likely between human photoreceptors and mouse retina? The triad ribbon synapse (TRS) that would normally form is unique and contains trans-synaptic proteins essential to its formation and function. Thus, could interspecific structural divergence be present that may inhibit this trans-synaptic bridge in discordant xenografts? In an effort to address this question computationally, we compared eight recently confirmed (including subcellular location) TRS specific (or predominantly expressed at the TRS) proteins among placental mammals (1-to-1 orthologs) using HyPhy selection analysis (a predictive measure of structural divergence) and by using Phyre2 tertiary structural modeling. Here, selection analysis revealed strong positive (diversifying) selection acting on a particularly important TRS protein: pikachurin. This positive selection was localized to its second Laminin-G (LG)-like domain and on its N-terminal domain – a putative region of trans-synaptic interaction. Localization of structural divergence to the N-terminus of each putative post-translational cleavage (PTC) product may suggest neofunctionalization from ancestral uncleaved pikachurin – this would be consistent with a recent counter-paradigm report of pikachurin cleavage predominating at the TRS. From this, we suggest a dual role after cleavage where the N-terminal fragment can still mediate the trans-synaptic bridge, while the C-terminal fragment may act as a diffusible trophic or “homing” factor for bipolar cell dendrite migration. Tertiary structural models mirrored the conformational divergence predicted by selection analysis. With human and mouse pikachurin (as well as other TRS proteins) likely to diverge considerably in structure among placental mammals – alongside known inter-mammalian variation in TRS phenotype and protein repertoire, high levels of diversifying selection acting on genes involving sensation, considerable timespans allowing for genetic drift that can create xenogeneic epistasis, and uncertainty surrounding the extent of xenosynaptogenesis in PPC transplant studies to date – use of distantly related hosts to test human photoreceptor graft therapeutic efficacy should be considered with caution.

Rare Synaptogenesis-Impairing Mutations in SLITRK5 Are Associated with Obsessive Compulsive Disorder.

Obsessive compulsive disorder (OCD) is substantially heritable, but few molecular genetic risk factors have been identified. Knockout mice lacking SLIT and NTRK-Like Family, Member 5 (SLITRK5) display OCD-like phenotypes including serotonin reuptake inhibitor-sensitive pathologic grooming, and corticostriatal dysfunction. Thus, mutations that impair SLITRK5 function may contribute to the genetic risk for OCD. We re-sequenced the protein-coding sequence of the human SLITRK5 gene (SLITRK5) in three hundred and seventy seven OCD subjects and compared rare non-synonymous mutations (RNMs) in that sample with similar mutations in the 1000 Genomes database. We also performed in silico assessments and in vitro functional synaptogenesis assays on the Slitrk5 mutations identified. We identified four RNM’s among these OCD subjects. There were no significant differences in the prevalence or in silico effects of rare non-synonymous mutations in the OCD sample versus controls. Direct functional testing of recombinant SLITRK5 proteins found that all mutations identified in OCD subjects impaired synaptogenic activity whereas none of the pseudo-matched mutations identified in 1000 Genomes controls had significant effects on SLITRK5 function (Fisher’s exact test P = 0.028). These results demonstrate that rare functional mutations in SLITRK5 contribute to the genetic risk for OCD in human populations. They also highlight the importance of biological characterization of allelic effects in understanding genotype-phenotype relationships as there were no statistical differences in overall prevalence or bioinformatically predicted effects of OCD case versus control mutations. Finally, these results converge with others to highlight the role of aberrant synaptic function in corticostriatal neurons in the pathophysiology of OCD.

Expressing Constitutively Active Rheb in Adult Dorsal Root Ganglion Neurons Enhances the Integration of Sensory Axons that Regenerate Across a Chondroitinase-Treated Dorsal Root Entry Zone Following Dorsal Root Crush.

While the peripheral branch of dorsal root ganglion neurons (DRG) can successfully regenerate after injury, lesioned central branch axons fail to regrow across the dorsal root entry zone (DREZ), the interface between the dorsal root and the spinal cord. This lack of regeneration is due to the limited regenerative capacity of adult sensory axons and the growth-inhibitory environment at the DREZ, which is similar to that found in the glial scar after a central nervous system (CNS) injury. We hypothesized that transduction of adult DRG neurons using adeno-associated virus (AAV) to express a constitutively-active form of the GTPase Rheb (caRheb) will increase their intrinsic growth potential after a dorsal root crush. Additionally, we posited that if we combined that approach with digestion of upregulated chondroitin sulfate proteoglycans (CSPG) at the DREZ with chondroitinase ABC (ChABC), we would promote regeneration of sensory axons across the DREZ into the spinal cord. We first assessed if this strategy promotes neuritic growth in an in vitro model of the glial scar containing CSPG. ChABC allowed for some regeneration across the once potently inhibitory substrate. Combining ChABC treatment with expression of caRheb in DRG significantly improved this growth. We then determined if this combination strategy also enhanced regeneration through the DREZ after dorsal root crush in adult rats in vivo. After unilaterally crushing C4-T1 dorsal roots, we injected AAV5-caRheb or AAV5-GFP into the ipsilateral C5-C8 DRGs. ChABC or PBS was injected into the ipsilateral dorsal horn at C5-C8 to digest CSPG, for a total of four animal groups (caRheb + ChABC, caRheb + PBS, GFP + ChABC, GFP + PBS). Regeneration was rarely observed in PBS-treated animals, whereas short-distance regrowth across the DREZ was observed in ChABC-treated animals. No difference in axon number or length between the ChABC groups was observed, which may be related to intraganglionic inflammation induced by the injection. ChABC-mediated regeneration is functional, as stimulation of ipsilateral median and ulnar nerves induced neuronal c-Fos expression in deafferented dorsal horn in both ChABC groups. Interestingly, caRheb + ChABC animals had significantly more c-Fos+ nuclei indicating that caRheb expression in DRGs promoted functional synaptogenesis of their axons that regenerated beyond a ChABC-treated DREZ.

Two classes of matrix metalloproteinases reciprocally regulate synaptogenesis

Synaptogenesis requires orchestrated intercellular communication between synaptic partners, with trans-synaptic signals necessarily traversing the extracellular synaptomatrix separating presynaptic and postsynaptic cells. Extracellular matrix metalloproteinases (Mmps) regulated by secreted tissue inhibitors of metalloproteinases (Timps), cleave secreted and membrane-associated targets to sculpt the extracellular environment and modulate intercellular signaling. Here, we test the roles of Mmp at the neuromuscular junction (NMJ) model synapse in the reductionist Drosophila system, which contains just two Mmps (secreted Mmp1 and GPI-anchored Mmp2) and one secreted Timp. We found that all three matrix metalloproteome components co-dependently localize in the synaptomatrix and show that both Mmp1 and Mmp2 independently restrict synapse morphogenesis and functional differentiation. Surprisingly, either dual knockdown or simultaneous inhibition of the two Mmp classes together restores normal synapse development, identifying a reciprocal suppression mechanism. The two Mmp classes co-regulate a Wnt trans-synaptic signaling pathway modulating structural and functional synaptogenesis, including the GPI-anchored heparan sulfate proteoglycan (HSPG) Wnt co-receptor Dally-like protein (Dlp), cognate receptor Frizzled-2 (Frz2) and Wingless (Wg) ligand. Loss of either Mmp1 or Mmp2 reciprocally misregulates Dlp at the synapse, with normal signaling restored by co-removal of both Mmp classes. Correcting Wnt co-receptor Dlp levels in both Mmp mutants prevents structural and functional synaptogenic defects. Taken together, these results identify an Mmp mechanism that fine-tunes HSPG co-receptor function to modulate Wnt signaling to coordinate synapse structural and functional development.

Synapsin knockdown is associated with decreased neurite outgrowth, functional synaptogenesis impairment, and fast high-frequency neurotransmitter release.

Synapsins (Syns) are an evolutionarily conserved family of synaptic vesicle-associated proteins related to fine tuning of synaptic transmission. Studies with mammals have partially clarified the different roles of Syns; however, the presence of different genes and isoforms and the development of compensatory mechanisms hinder accurate data interpretation. Here, we use a simple in vitro monosynaptic Helix neuron connection, reproducing an in vivo physiological connection as a reliable experimental model to investigate the effects of Syn knockdown. Cells overexpressing an antisense construct against Helix Syn showed a time-dependent decrease of Syn immunostaining, confirming protein loss. At the morphological level, Syn-silenced cells showed a reduction in neurite linear outgrowth and branching and in the size and number of synaptic varicosities. Functionally, Syn-silenced cells presented a reduced ability to form synaptic connections; however, functional chemical synapses showed similar basal excitatory postsynaptic potentials and similar short-term plasticity paradigms. In addition, Syn-silenced cells presented faster neurotransmitter release and decreased postsynaptic response toward the end of long tetanic presynaptic stimulations, probably related to an impairment of the synaptic vesicle trafficking resulting from a different vesicle handling, with an increased readily releasable pool and a compromised reserve pool.

Two classes of matrix metalloproteinases reciprocally regulate synaptogenesis.

Synaptogenesis requires orchestrated intercellular communication between synaptic partners, with trans-synaptic signals necessarily traversing the extracellular synaptomatrix separating presynaptic and postsynaptic cells. Extracellular matrix metalloproteinases (Mmps) regulated by secreted tissue inhibitors of metalloproteinases (Timps), cleave secreted and membrane-associated targets to sculpt the extracellular environment and modulate intercellular signaling. Here, we test the roles of Mmp at the neuromuscular junction (NMJ) model synapse in the reductionist Drosophila system, which contains just two Mmps (secreted Mmp1 and GPI-anchored Mmp2) and one secreted Timp. We found that all three matrix metalloproteome components co-dependently localize in the synaptomatrix and show that both Mmp1 and Mmp2 independently restrict synapse morphogenesis and functional differentiation. Surprisingly, either dual knockdown or simultaneous inhibition of the two Mmp classes together restores normal synapse development, identifying a reciprocal suppression mechanism. The two Mmp classes co-regulate a Wnt trans-synaptic signaling pathway modulating structural and functional synaptogenesis, including the GPI-anchored heparan sulfate proteoglycan (HSPG) Wnt co-receptor Dally-like protein (Dlp), cognate receptor Frizzled-2 (Frz2) and Wingless (Wg) ligand. Loss of either Mmp1 or Mmp2 reciprocally misregulates Dlp at the synapse, with normal signaling restored by co-removal of both Mmp classes. Correcting Wnt co-receptor Dlp levels in both Mmp mutants prevents structural and functional synaptogenic defects. Taken together, these results identify an Mmp mechanism that fine-tunes HSPG co-receptor function to modulate Wnt signaling to coordinate synapse structural and functional development.

Importance of being Nernst: Synaptic activity and functional relevance in stem cell-derived neurons.

Functional synaptogenesis and network emergence are signature endpoints of neurogenesis. These behaviors provide higher-order confirmation that biochemical and cellular processes necessary for neurotransmitter release, post-synaptic detection and network propagation of neuronal activity have been properly expressed and coordinated among cells. The development of synaptic neurotransmission can therefore be considered a defining property of neurons. Although dissociated primary neuron cultures readily form functioning synapses and network behaviors in vitro, continuously cultured neurogenic cell lines have historically failed to meet these criteria. Therefore, in vitro-derived neuron models that develop synaptic transmission are critically needed for a wide array of studies, including molecular neuroscience, developmental neurogenesis, disease research and neurotoxicology. Over the last decade, neurons derived from various stem cell lines have shown varying ability to develop into functionally mature neurons. In this review, we will discuss the neurogenic potential of various stem cells populations, addressing strengths and weaknesses of each, with particular attention to the emergence of functional behaviors. We will propose methods to functionally characterize new stem cell-derived neuron (SCN) platforms to improve their reliability as physiological relevant models. Finally, we will review how synaptically active SCNs can be applied to accelerate research in a variety of areas. Ultimately, emphasizing the critical importance of synaptic activity and network responses as a marker of neuronal maturation is anticipated to result in in vitro findings that better translate to efficacious clinical treatments.

Post Ischemia Intermittent Hypoxia Induces Hippocampal Neurogenesis and Synaptic Alterations and Alleviates Long-Term Memory Impairment

Adult hippocampal neurogenesis is important for learning and memory, especially after a brain injury such as ischemia. Newborn hippocampal neurons contribute to memory performance by establishing functional synapses with target cells. This study demonstrated that the maturation of hippocampal neurons is enhanced by postischemia intermittent hypoxia (IH) intervention. The effects of IH intervention in cultured neurons were mediated by increased synaptogenesis, which was primarily regulated by brain-derived neurotrophic factor (BDNF)/PI3K/AKT. Hippocampal neo-neurons expressed BDNF and exhibited enhanced presynaptic function as indicated by increases in the pSynapsin expression, synaptophysin intensity, and postsynapse density following IH intervention after ischemia. Postischemia IH-induced hippocampal neo-neurons were affected by presynaptic activity, which reflected the dynamic plasticity of the glutamatergic receptors. These alterations were also associated with the alleviation of ischemia-induced long-term memory impairment. Our results suggest that postischemia IH intervention rescued ischemia-induced spatial learning and memory impairment by inducing hippocampal neurogenesis and functional synaptogenesis via BDNF expression.

Single-sided deafness leads to unilateral aural preference within an early sensitive period

Unilateral deafness has a high incidence in children. In addition to children who are born without hearing in one ear, children with bilateral deafness are frequently equipped only with one cochlear implant, leaving the other ear deaf. The present study investigates the effects of such single-sided deafness during development in the congenitally deaf cat. The investigated animals were either born with unilateral deafness or received a cochlear implant in one ear and were subjected to chronic monaural stimulation. In chronically stimulated animals, implantation ages were at the following three critical developmental points: 'early' during the peak of functional cortical synaptogenesis in deaf animals; 'intermediate' at the age when synaptic activity in the deaf cats dropped to the level of hearing control cats and finally, 'late' at the age when the evoked synaptic activity fell below the level of hearing control cats. After periods of unilateral hearing, local field potentials were recorded from the cortical surface using a microelectrode at ∼100 recording positions. Stimulation was with cochlear implants at both ears. The measures evaluated were dependent only on the symmetry of aural input: paired differences of onset latencies and paired relations of peak amplitudes of local field potentials. A massive reorganization of aural preference in favour of the hearing ear was found in these measures if the onset of unilateral hearing was early (before or around the peak of functional synaptogenesis). The effect was reduced if onset of unilateral hearing was in the intermediate period, and it disappeared if the onset was late. In early onset of unilateral deafness, the used ear became functionally dominant with respect to local field potential onset latency and amplitude. This explains the inferior outcome of implantations at the second-implanted ear compared with first-implanted ear in children. However, despite a central disadvantage for the deaf ear, it still remained capable of activating the auditory cortex. Appropriate training may thus help to improve the performance at the second-implanted ear. In conclusion, periods of monaural stimulation should be kept as short as possible, and training focused on the deaf ear should be introduced after delayed second implantation in children.

Functional complementation of hippocampus with exogenous neural stem cells for cognitive recovery after application of kainic acid

As photoreceptor transplantation rapidly moves closer to the clinic, verifying graft efficacy in animal models may have unforeseen xenogeneic barriers. Although photoreceptor transplants have most convincingly exhibited functional synaptogenesis in conspecific studies, such evidence (while ruling out false-positives due to: viral graft labeling, fusion/cytosolic transfer, or neuroprotection) has not yet been shown for discordant xenografts. From this, a fundamental question should be raised: is useful xenosynaptogenesis likely between human photoreceptors and mouse retina? The triad ribbon synapse (TRS) that would normally form is unique and contains trans-synaptic proteins essential to its formation and function. Thus, could interspecific structural divergence be present that may inhibit this trans-synaptic bridge in discordant xenografts? In an effort to address this question computationally, we compared eight recently confirmed (including subcellular location) TRS specific (or predominantly expressed at the TRS) proteins among placental mammals (1-to-1 orthologs) using HyPhy selection analysis (a predictive measure of structural divergence) and by using Phyre2 tertiary structural modeling. Here, selection analysis revealed strong positive (diversifying) selection acting on a particularly important TRS protein: pikachurin. This positive selection was localized to its second Laminin-G (LG)-like domain and on its N-terminal domain – a putative region of trans-synaptic interaction. Localization of structural divergence to the N-terminus of each putative post-translational cleavage (PTC) product may suggest neofunctionalization from ancestral uncleaved pikachurin – this would be consistent with a recent counter-paradigm report of pikachurin cleavage predominating at the TRS. From this, we suggest a dual role after cleavage where the N-terminal fragment can still mediate the trans-synaptic bridge, while the C-terminal fragment may act as a diffusible trophic or “homing” factor for bipolar cell dendrite migration. Tertiary structural models mirrored the conformational divergence predicted by selection analysis. With human and mouse pikachurin (as well as other TRS proteins) likely to diverge considerably in structure among placental mammals – alongside known inter-mammalian variation in TRS phenotype and protein repertoire, high levels of diversifying selection acting on genes involving sensation, considerable timespans allowing for genetic drift that can create xenogeneic epistasis, and uncertainty surrounding the extent of xenosynaptogenesis in PPC transplant studies to date – use of distantly related hosts to test human photoreceptor graft therapeutic efficacy should be considered with caution.

Phosphoinositide-3-kinase activation controls synaptogenesis and spinogenesis in hippocampal neurons.

The possibility of changing the number of synapses may be an important asset in the treatment of neurological diseases. In this context, the synaptogenic role of the phosphoinositide-3-kinase (PI3K) signaling cascade has been previously demonstrated in Drosophila. This study shows that treatment with a PI3K-activating transduction peptide is able to promote synaptogenesis and spinogenesis in primary cultures of rat hippocampal neurons, as well as in CA1 hippocampal neurons in vivo. In culture, the peptide increases synapse density independently of cell density, culture age, dendritic complexity, or synapse type. The induced synapses also increase neurotransmitter release from cultured neurons. The synaptogenic signaling pathway includes PI3K-Akt. Furthermore, the treatment is effective on adult neurons, where it induces spinogenesis and enhances the cognitive behavior of treated animals in a fear-conditioning assay. These findings demonstrate that functional synaptogenesis can be induced in mature mammalian brains through PI3K activation.

The Cdc42-selective GAP Rich regulates postsynaptic development and retrograde BMP transsynaptic signaling

Retrograde bone morphogenetic protein signaling mediated by the Glass bottom boat (Gbb) ligand modulates structural and functional synaptogenesis at the Drosophila melanogaster neuromuscular junction. However, the molecular mechanisms regulating postsynaptic Gbb release are poorly understood. In this study, we show that Drosophila Rich (dRich), a conserved Cdc42-selective guanosine triphosphatase-activating protein (GAP), inhibits the Cdc42-Wsp pathway to stimulate postsynaptic Gbb release. Loss of dRich causes synaptic undergrowth and strongly impairs neurotransmitter release. These presynaptic defects are rescued by targeted postsynaptic expression of wild-type dRich but not a GAP-deficient mutant. dRich inhibits the postsynaptic localization of the Cdc42 effector Wsp (Drosophila orthologue of mammalian Wiskott-Aldrich syndrome protein, WASp), and manifestation of synaptogenesis defects in drich mutants requires Wsp signaling. In addition, dRich regulates postsynaptic organization independently of Cdc42. Importantly, dRich increases Gbb release and elevates presynaptic phosphorylated Mad levels. We propose that dRich coordinates the Gbb-dependent modulation of synaptic growth and function with postsynaptic development.

A novel approach reveals temporal patterns of synaptogenesis between the isolated growth cones of Lymnaea neurons

All brain functions, ranging from motor behaviour to cognition, depend on precise developmental patterns of synapse formation between the growth cones of both pre- and postsynaptic neurons. While the molecular evidence for the presence of 'pre-assembled' elements of synaptic machinery prior to physical contact is beginning to emerge, the precise timing of functional synaptogenesis between the growth cones has not yet been defined. Moreover, it is unclear whether an initial assembly of various synaptic molecules located at the extrasomal regions (e.g. growth cones) can indeed result in fully mature and consolidated synapses in the absence of somata signalling. Such evidence is difficult to obtain both in vivo and in vitro because the extrasomal sites are often challenging, if not impossible, to access for electrophysiological analysis. Here we demonstrate a novel approach to precisely define various steps underlying synapse formation between the isolated growth cones of individually identifiable pre- and postsynaptic neurons from the mollusc Lymnaea stagnalis. We show for the first time that isolated growth cones transformed into 'growth balls' have an innate propensity to develop specific and multiple synapses within minutes of physical contact. We also demonstrate that a prior 'synaptic history' primes the presynaptic growth ball to form synapses quicker with subsequent partners. This is the first demonstration that isolated Lymnaea growth cones have the necessary machinery to form functional synapses.

Genetic controls balancing excitatory and inhibitory synaptogenesis in neurodevelopmental disorder models.

Proper brain function requires stringent balance of excitatory and inhibitory synapse formation during neural circuit assembly. Mutation of genes that normally sculpt and maintain this balance results in severe dysfunction, causing neurodevelopmental disorders including autism, epilepsy and Rett syndrome. Such mutations may result in defective architectural structuring of synaptic connections, molecular assembly of synapses and/or functional synaptogenesis. The affected genes often encode synaptic components directly, but also include regulators that secondarily mediate the synthesis or assembly of synaptic proteins. The prime example is Fragile X syndrome (FXS), the leading heritable cause of both intellectual disability and autism spectrum disorders. FXS results from loss of mRNA-binding FMRP, which regulates synaptic transcript trafficking, stability and translation in activity-dependent synaptogenesis and plasticity mechanisms. Genetic models of FXS exhibit striking excitatory and inhibitory synapse imbalance, associated with impaired cognitive and social interaction behaviors. Downstream of translation control, a number of specific synaptic proteins regulate excitatory versus inhibitory synaptogenesis, independently or combinatorially, and loss of these proteins is also linked to disrupted neurodevelopment. The current effort is to define the cascade of events linking transcription, translation and the role of specific synaptic proteins in the maintenance of excitatory versus inhibitory synapses during neural circuit formation. This focus includes mechanisms that fine-tune excitation and inhibition during the refinement of functional synaptic circuits, and later modulate this balance throughout life. The use of powerful new genetic models has begun to shed light on the mechanistic bases of excitation/inhibition imbalance for a range of neurodevelopmental disease states.

Increase of Kv3.1b expression in avian auditory brainstem neurons correlates with synaptogenesis in vivo and in vitro

In the auditory system voltage-activated currents mediated by potassium channels Kv1.1 and Kv3.1b and their interaction with sodium inward currents play a crucial role for computational function. However, it is unresolved how these potassium channels are developmentally regulated. We have therefore combined a biochemical investigation of Kv1.1 and Kv3.1b protein expression with electrophysiological recordings of membrane currents to characterize neuronal differentiation in the auditory brain stem of the chick. Differentiation in vitro was compared with cells prepared from corresponding embryonic stages in vivo. Using a computer model based on the empirical data we were then able to predict physiological properties of developing auditory brain stem neurons. In vivo Kv3.1b expression increased strongly between E10 and E14, a time of functional synaptogenesis in the auditory brainstem. We also found this increase of expression in vitro, again coinciding with synaptogenesis in the cultures. Whole-cell patch recordings revealed a corresponding increase of the (Kv3.1-like) high threshold potassium current. In contrast, Kv1.1 protein expression failed to increase in vitro, and changes in (Kv1.1-like) low threshold potassium current with time in culture were not significant. Electrophysiological recordings revealed that sodium inward currents increased with cultivation time. Thus, our data suggest that Kv3.1b expression occurs with the onset of functional synaptogenesis, while a different signal, absent from cultures of dissociated auditory brain stem, is needed for Kv1.1 expression. A biophysical model constructed with parameters from our recordings was used to investigate the functional impact of the currents mediated by these channels. We found that during development both high and low threshold potassium currents need to be increased in a concerted manner with the sodium conductance for the neurons to exhibit fast and phasic action potential firing and a narrow time window of coincidence detection.

Neuronal differentiation of the early embryonic auditory hindbrain of the chicken in primary culture

Neurons in the auditory hindbrain pathway of the chicken are physiologically and morphologically highly specialized. It remains unclear to what extent independent differentiation vs. activity-dependent mechanisms determines the development of this system. To address this question we established a primary culture system of the early auditory hindbrain neurons. Primary cultures of neurons from nucleus magnocellularis and nucleus laminaris were prepared from embryonic day 6.5 chicken. These cells developed in culture under serum-free conditions for up to 15 days. Immunocytochemical staining and whole-cell patch recordings were used to characterize the development of the neurons. A stable expression of the calcium-binding protein calretinin, which serves as a characteristic marker of the auditory pathway, was found at all stages. A voltage-gated potassium channel (Kv3.1b) with a specific function in the auditory system was also expressed after about 1 week in culture. Electrophysiological recordings showed a general maturation of the neuronal phenotype as reflected by an increase in the mean resting membrane potential, a decrease in the mean input resistance as well as a maturation of action potential parameters. Four groups of neurons that generate action potentials could be distinguished. One of these showed the phasic firing pattern of auditory brainstem neurons known from slice preparations. In older cultures we demonstrated functional synaptogenesis in vitro by recording postsynaptic activity elicited by extracellular stimulation and styryl dye loading of vesicles. Thus, isolated neurons from the auditory region of the avian brainstem differentiate to specific neuronal subtypes and autonomously develop synaptic connections in vitro.

Expression of MeCP2 in olfactory receptor neurons is developmentally regulated and occurs before synaptogenesis

Rett syndrome, a neurodevelopmental disorder hypothesized to be due to defective neuronal maturation, is a result of mutations in the mecp2 gene encoding the transcriptional repressor methyl-CpG binding protein (MeCP2). We utilized the olfactory system, which displays postnatal neurogenesis, as a model to investigate MeCP2 expression during development and after injury. MeCP2 expression increased postnatally, localizing to mature olfactory receptor neurons (ORNs) and sustentacular supporting cells. The timing of MeCP2 expression was defined by using detergent ablation (to remove the ORNs) and unilateral olfactory bulbectomy (to remove the ORN target), both of which increase neurogenesis. MeCP2 expression in the ORNs reached prelesioning levels as cells matured after ablation, whereas expression was not completely restored after bulbectomy, in which functional synaptogenesis cannot occur. Thus, MeCP2 expression correlates with the maturational state of ORNs, and precedes synaptogenesis. Identifying the time window of MeCP2 expression should help further clarify the biological defects in Rett syndrome.

Development of convergent synaptic inputs to subpopulations of autonomic neurons.

Visceromotor neurons in mammalian prevertebral sympathetic ganglia receive convergent synaptic inputs from spinal preganglionic neurons and peripheral intestinofugal neurons projecting from the enteric plexuses. Vasomotor neurons in the same ganglia receive only preganglionic inputs. How this pathway-specific pattern of connectivity is established is unknown. We have used a combination of immunohistochemical, ultrastructural, and electrophysiological techniques to investigate the development of synaptic inputs onto visceromotor and vasomotor neurons in the celiac ganglion of guinea pigs. Functional synaptogenesis occurred primarily from early fetal (F30-F35) to midfetal (F36-F45) stages, after the neurochemical differentiation of vasomotor and visceromotor neurons but before establishment of their electrophysiological phenotypes. Intestinofugal inputs were detected only on presumptive visceromotor neurons located primarily in medial regions of the ganglion. The number of ultrastructurally identified synaptic profiles increased in parallel with functional synaptogenesis, especially in medial regions, where dendritic growth rates also were higher. However, the expression of immunoreactivity to choline acetyltransferase in the terminals of inputs was very low until late fetal stages, after functional transmission already had been established. These results show that peripheral intestinofugal neurons directly establish appropriate functional connections with their target visceromotor neurons simultaneously with the development of functional preganglionic inputs to both visceromotor and vasomotor neurons. It seems likely that synaptogenesis occurs independently of the neurochemical differentiation of the target neurons but is closely related to the pathway-specific dendritic development of those neurons.