Activity-dependent changes in synaptic efficacy at glutamatergic and GABAergic connections in the immature hippocampus

During development, correlated network activity plays a crucial role in establishing functional synaptic contacts, thus contributing to the development of adult neuronal circuits. In the hippocampus, a region of the brain involved in the formation of declarative memories, network-driven giant depolarizing potentials or GDPs are generated by the synergistic action of glutamate and GABA, which at this developmental stage is depolarizing and excitatory. The depolarizing action of GABA during GDPs results in calcium flux via NMDA receptors and voltage-dependent calcium channels. Calcium, in turn activates different signaling pathways necessary for several developmental processes including synaptogenesis. In previous work from our laboratory it was demonstrated that GDPs and associated calcium transients act as coincident detectors for enhancing synaptic efficacy at poorly developed mossy fibre-CA3 synapses in a Hebbian type of way..

Plasticity of GABA(B) receptor-mediated heterosynaptic interactions at mossy fibers after status epilepticus

Several neurotransmitters, including GABA acting at presynaptic GABAB receptors, modulate glutamate release at synapses between hippocampal mossy fibers and CA3 pyramidal neurons. This phenomenon gates excitation of the hippocampus and may therefore prevent limbic seizure propagation. Here we report that status epilepticus, triggered by either perforant path stimulation or pilocarpine administration, was followed 24 hr later by a loss of GABAB receptor-mediated heterosynaptic depression among populations of mossy fibers. This was accompanied by a decrease in the sensitivity of mossy fiber transmission to the exogenous GABAB receptor agonist baclofen. Autoradiography revealed a reduction in GABAB receptor binding in the stratum lucidum after status epilepticus. Failure of GABAB receptor-mediated modulation of mossy fiber transmission at mossy fibers may contribute to the development of spontaneous seizures after status epilepticus

The Mammalian Interaural Time Difference Detection Circuit Is Differentially Controlled by GABAB Receptors during Development

Throughout development GABAB receptors (GABABRs) are widely expressed in the mammalian brain. In mature auditory brainstem neurons, GABABRs are involved in the short-term regulation of the strength and dynamics of excitatory and inhibitory inputs, thus modulating sound analysis. During development, GABABRs also contribute to long-term changes in input strength. Using a combination of whole-cell patch-clamp recordings in acute brain slices and immunostainings in gerbils, we characterized developmental changes in GABABR-mediated regulation of synaptic inputs to neurons in the medial superior olive (MSO), an auditory brainstem nucleus that analyzes interaural time differences (ITDs). Here, we show that, before hearing onset, GABABR-mediated depression of transmitter release is much stronger for excitation than inhibition, whereas in mature animals GABABRs mainly control the inhibition. During the same developmental period, GABABR immunoreactivity shifts from the dendritic to the somatic region of the MSO. Furthermore, only before hearing onset (postnatal day 12), stimulation of the fibers originating in the medial and the lateral nucleus of the trapezoid body (MNTB and LNTB) activates GABABRs on both the inhibitory and the excitatory inputs. After hearing onset, GAD65-positive endings devoid of glycine transporter reactivity suggest GABA release from sources other than the MNTB and LNTB. At this age, pharmacological increase of spontaneous synaptic release activates GABABRs only on the inhibitory inputs. This indicates not only a profound inhibitory effect of GABABRs on the major inputs to MSO neurons in neonatal animals but also a direct modulatory role of GABABRs for ITD analysis in the MSO of adult animals

Activity-dependent regulation of GABA release at immature mossy fibers-CA3 synapses: role of the Prion protein

In adulthood, mossy fibers (MFs), the axons of granule cells of the dentate gyrus (DG), release glutamate onto CA3 principal cells and interneurons. In contrast, during the first week of postnatal life MFs release -aminobutyric acid (GABA), which, at this early developmental stage exerts a depolarizing and excitatory action on targeted cells. The depolarizing action of GABA opens voltage-dependent calcium channels and NMDA receptors leading to calcium entry and activation of intracellular signaling pathways involved in several developmental processes, thus contributing to the refinement of neuronal connections and to the establishment of adult neuronal circuits. The release of GABA has been shown to be down regulated by several neurotransmitter receptors which would limit the enhanced excitability caused by the excitatory action of GABA. It is worth noting that the immature hippocampus exhibits spontaneous correlated activity, the so called giant depolarizing potentials or GDPs that act as coincident detector signals for enhancing synaptic activity, thus contributing to several developmental processes including synaptogenesis. GDPs render the immature hippocampus more prone to seizures. Here, I explored the molecular mechanisms underlying synaptic transmission and activity-dependent synaptic plasticity processes at immature GABAergic MF-CA3 synapses in wild-type rodents and in mice lacking the prion protein (Prnp0/0 mice). In the first paper, I studied the functional role of kainate receptors (KARs) in regulating GABA release from MF terminals. Presynaptic KARs regulate synaptic transmission in several brain areas and play a central role in modulating glutamate release at adult MF-CA3 synapses. I found that functional presynaptic GluK1 receptors are present on MF terminals where they down regulate GABA release. Thus, application of DNQX or UBP 302, a selective antagonist for GluK1 receptors, strongly increased the amplitude of MF-GABAA-mediated postsynaptic currents (GPSCs). This effect was associated with a decrease in failure rate and increase in PPR, indicating a presynaptic type of action. GluK1 receptors were found to be tonically activated by glutamate present in the extracellular space, since decreasing the extracellular concentration of glutamate with a glutamate scavenger system prevented their activation and mimicked the effects of KAR antagonists. The depressant effect of GluK1 on GABA release was dependent on pertussis toxin (PTx)-sensitive G protein-coupled kainate receptors since it was prevented when hippocampal slices were incubated in the presence of a solution containing PTx. This effect was presynaptic since application of UBP 302 to cells patched with an intracellular solution containing GDP S still potentiated synaptic responses. In addition, the depressant effect of GluK1 on GABA release was prevented by U73122, which selectively inhibits phospholipase C, downstream to G protein activation. Interestingly, U73122, enhanced the probability of GABA release, thus unveiling the ionotropic type of action of kainate receptors. In line with this, we found that GluK1 receptors enhanced MF excitability by directly depolarizing MF terminals via calcium-permeable cation channels. We also explored the possible involvement of GluK1 in spike time-dependent (STD) plasticity and we found that GluK1 dynamically regulate the direction of STD-plasticity, since the pharmacological block of this receptor shifted spike-time dependent potentiation into depression. The mechanisms underlying STD-LTD at immature MF-CA3 synapses have been investigated in detail in the second paper. STD-plasticity is a Hebbian form of learning which consists in bi-directional modifications of synaptic strength according to the temporal order of pre and postsynaptic spiking. Interestingly, we found that at immature mossy fibers (MF)-CA3 synapses, STD-LTD occurs regardless of the temporal order of stimulation (pre versus post or viceversa). However, as already mentioned, while STD-LTD induced by positive pairing (pre before post) could be shifted into STD-LTP after blocking presynaptic GluK1 receptors, STD-LTD induced by negative pairing (post before pre) relied on the activation of CB1 receptors. At P3 but not at P21, endocannabinoids released by the postsynaptic cell during spiking-induced membrane depolarization retrogradely activated CB1 receptors, probably expressed on MF terminals and persistently depressed GABA release in the rat hippocampus. Thus, bath application of selective CB1 receptor antagonists prevented STD-LTD. Pharmacological tools allow identifying anandamide as the endogenous ligand responsible of activity-dependent depressant effect. To further assess whether STD-LTD is dependent on the activation of CB1 receptors, similar experiments were performed on WT-littermates and CB1-KO mice. While in WT mice the pairing protocol produced a persistent depression of MF-GPSCs as in rats, in CB1-KO mice failed to induce LTD. Consistent with these data, in situ hybridization experiments revealed detectable levels of CB1 mRNA in the granule cell layer of P3 but not of P21mice. These experiments strongly suggest that at immature MF-CA3 synapses STD-LTD is mediated by CB1 receptors, probably transiently expressed, during a critical time window, on MF terminals. In the third paper, I studied synaptic transmission and activity dependent synaptic plasticity at immature MF-CA3 synapses in mice devoid of the prion protein (Prnp0/0). The prion protein (PrPC) is a conserved glycoprotein widely expressed in the brain and involved in several neuronal processes including neurotransmission. If converted to a conformationally altered form, PrPSc can cause neurodegenerative diseases, such as Creutzfeldt-Jakob disease in humans. Previous studies aimed at characterizing Prnp0/0 mice have revealed only mild behavioral changes, including an impaired spatial learning, accompanied by electrophysiological and biochemical alterations. Interestingly, PrPC is developmentally regulated and in the hippocampus its expression parallels the maturation of MF. Here, we tested the hypothesis that at immature (P3-P7) MF-CA3 synapses, PrPC interferes with synaptic plasticity processes. To this aim, the rising phase of Giant Depolarizing Potentials (GDPs), a hallmark of developmental networks, was used to stimulate granule cells in the dentate gyrus in such a way that GDPs were coincident with afferent inputs. In WT animals, the pairing procedure induced a persistent increase in amplitude of MF-GPSCs. In contrast, in Prnp0/0 mice, the same protocol produced a long-term depression (LTD). LTP was postsynaptic in origin and required the activation of cAMP-dependent PKA signaling while LTD was presynaptic and was reliant on G protein-coupled GluK1 receptor and protein lipase C downstream to G protein activation. In addition, at emerging CA3-CA1 synapses of PrPC-deficient mice, stimulation of Schaffer collateral failed to induce LTP, known to be PKA-dependent. Finally, we also found that LTD in Prnp0/0 mice was mediated by GluK1 receptors, since UBP 302 blocked its induction. These data suggest that in the immature hippocampus PrPC controls the direction of synaptic plasticity

Strategically Positioned Inhibitory Synapses of Axo-axonic Cells Potently Control Principal Neuron Spiking in the Basolateral Amygdala.

Axo-axonic cells (AACs) in cortical regions selectively innervate the axon initial segments (AISs) of principal cells (PCs), where the action potentials are generated. These GABAergic interneurons can alter the activity of PCs, but how the efficacy of spike control correlates with the number of output synapses remains unclear. Moreover, the relationship between the spatial distribution of GABAergic synapses and the action potential initiation site along the AISs is not well defined. Using paired recordings obtained in the mouse basolateral amygdala, we found that AACs powerfully inhibited or delayed the timing of PC spiking by 30 ms, if AAC output preceded PC spiking with no more than 80 ms. By correlating the number of synapses and the probability of spiking, we revealed that larger numbers of presynaptic AAC boutons giving rise to larger postsynaptic responses provided more effective inhibition of PC spiking. At least 10-12 AAC synapses, which could originate from 2-3 AACs on average, were necessary to veto the PC firing under our recording conditions. Furthermore, we determined that the threshold for the action potential generation along PC axons is the lowest between 20 and 40 mum from soma, which axonal segment received the highest density of GABAergic inputs. Single AACs preferentially innervated this narrow portion of the AIS where action potentials were generated with the highest likelihood, regardless of the number of synapses forming a given connection. Our results uncovered a fine organization of AAC innervation maximizing their inhibitory efficacy by strategically positioning synapses along the AISs

Signalling properties at single synapses and within the interneuronal network in the CA1 region of the rodent hippocampus

Understanding how the complexity of connections among the neurons in the brain is established and modified in an experience- and activity-dependent way is a challenging task of Neuroscience. Although in the last decades many progresses have been made in characterising the basic mechanisms of synaptic transmission, a full comprehension of how information is transferred and processed by neurons has not been fully achieved. In the present study, theoretical tools and patch clamp experiments were used to further investigate synaptic transmission, focusing on quantal transmission at single synapses and on different types of signalling at the level of a particular interneuronal network in the CA1 area of the rodent hippocampus. The simultaneous release of more than one vesicle from an individual presynaptic active zone is a typical mechanism that can affect the strength and reliability of synaptic transmission. At many central synapses, however, release caused by a single presynaptic action potential is limited to one vesicle (univesicular release). The likelihood of multivesicular release at a particular synapse has been tied to release probability (Pr), and whether it can occur at Schaffer collateral\u2013CA1 synapses, at which Pr ranges widely, is controversial. In contrast with previous findings, proofs of multivesicular release at this synapse have been recently obtained at late developmental stages; however, in the case of newborn hippocampus, it is still difficult to find strong evidence in one direction or another. In order to address this point, in the first part of this study a simple and general stochastic model of synaptic release has been developed and analytically solved. The model solution gives analytical mathematical expressions relating basic quantal parameters with average values of quantities that can be measured experimentally. Comparison of these quantities with the experimental measures allows to determine the most probable values of the quantal parameters and to discriminate the univesicular from the multivesicular mode of glutamate release. The model has been validated with data previously collected at glutamatergic CA3-CA1 synapses in the hippocampus from newborn (P1-P5 old) rats. The results strongly support a multivesicular type of release process requiring a variable pool of immediately releasable vesicles. Moreover, computing quantities that are functions of the model parameters, the mean amplitude of the synaptic response to the release of a single vesicle (Q) was estimated to be 5-10 pA, in very good agreement with experimental findings. In addition, a multivesicular type of release was supported by various experimental evidences: a high variability of the amplitude of successes, with a coefficient of variation ranging from 0.12 to 0.73; an average potency ratio a2/a1 between the second and first response to a pair of stimuli bigger than 1; and changes in the potency of the synaptic response to the first stimulus when the release probability was modified by increasing or decreasing the extracellular calcium concentration. This work indicates that at glutamatergic CA3-CA1 synapses of the neonatal rat hippocampus a single action potential may induce the release of more than one vesicle from the same release site. In a more systemic approach to the analysis of communication between neurons, it is interesting to investigate more complex, network interactions. GABAergic interneurons constitute a heterogeneous group of cells which exert a powerful control on network excitability and are responsible for the oscillatory behaviour crucial for information processing in the brain. They have been differently classified according to their morphological, neurochemical and physiological characteristics. In the second part of this study, whole cell patch clamp recordings were used to further characterize, in transgenic mice expressing EGFP in a subpopulation of GABAergic interneurons containing somatostatin (GIN mice), the functional properties of EGFPpositive cells in stratum oriens of the CA1 region of the hippocampus, in slice cultures obtained from P8 old animals. These cells showed passive and active membrane properties similar to those found in stratum oriens interneurons projecting to stratum lacunosum-moleculare. Moreover, they exhibited different firing patterns which were maintained upon membrane depolarization: irregular (48%), regular (30%) and clustered (22%). Paired recordings from EGFP-positive cells often revealed electrical coupling (47% of the cases), which was abolished by carbenoxolone (200 mM). On average, the coupling coefficient was 0.21 \ub1 0.07. When electrical coupling was particularly strong it acted as a powerful low-pass filter, thus contributing to alter the output of individual cells. The dynamic interaction between cells with various firing patterns may differently control GABAergic signalling, leading, as suggested by simulation data, to a wide range of interneuronal communication. In additional paired recordings of a presynaptic EGFP positive interneuron and a postsynaptic principal cell, trains of action potentials in interneurons rarely evoked GABAergic postsynaptic currents (3/45 pairs) with small amplitude and slow kinetics, and that at 20 Hz exhibited short-term depression. In contrast, excitatory connections between principal cells and EGFP-positive interneurons were found more often (17/55 pairs) and exhibited a frequency and use-dependent facilitation, particularly in the gamma band. In conclusion, it appears that EGFP-positive interneurons in stratum oriens of GIN mice constitute a heterogeneous population of cells interconnected via electrical synapses, exhibiting particular features in their chemical and electrical synaptic signalling. Moreover, the dynamic interaction between these interneurons may differentially affect target cells and neuronal communication within the hippocampal network

GABAergic Activities Control Spike Timing- and Frequency-Dependent Long-Term Depression at Hippocampal Excitatory Synapses

GABAergic interneuronal network activities in the hippocampus control a variety of neural functions, including learning and memory, by regulating θ and γ oscillations. How these GABAergic activities at pre- and postsynaptic sites of hippocampal CA1 pyramidal cells differentially contribute to synaptic function and plasticity during their repetitive pre- and postsynaptic spiking at θ and γ oscillations is largely unknown. We show here that activities mediated by postsynaptic GABAARs and presynaptic GABABRs determine, respectively, the spike timing- and frequency-dependence of activity-induced synaptic modifications at Schaffer collateral-CA1 excitatory synapses. We demonstrate that both feedforward and feedback GABAAR-mediated inhibition in the postsynaptic cell controls the spike timing-dependent long-term depression of excitatory inputs (“e-LTD”) at the θ frequency. We also show that feedback postsynaptic inhibition specifically causes e-LTD of inputs that induce small postsynaptic currents (<70 pA) with LTP-timing, thus enforcing the requirement of cooperativity for induction of long-term potentiation at excitatory inputs (“e-LTP”). Furthermore, under spike-timing protocols that induce e-LTP and e-LTD at excitatory synapses, we observed parallel induction of LTP and LTD at inhibitory inputs (“i-LTP” and “i-LTD”) to the same postsynaptic cells. Finally, we show that presynaptic GABABR-mediated inhibition plays a major role in the induction of frequency-dependent e-LTD at α and β frequencies. These observations demonstrate the critical influence of GABAergic interneuronal network activities in regulating the spike timing- and frequency-dependences of long-term synaptic modifications in the hippocampus

Trans-synaptic signaling at GABAergic connections: possible dysfunction in some forms of Autism Spectrum Disorders

Synapses are recognized as being highly plastic in structure and function, strongly influenced by their own histories of impulse traffic and by signals from nearby cells. Synaptic contacts are fundamental for the development, homeostasis and remodeling of complex neural circuits. Synapses are highly varied in their molecular composition. Understand this diversity is important because it sheds light on the way they function. In particular, this may be useful for understanding the mechanisms at the basis of synaptic dysfunctions associated with neurodevelopmental disorders, such as Autism Spectrum Disorders (ASD) in order to develop properly targeted therapeutic tools. During the first part of my Phd course I characterized the functional role of gephyrin at inhibitory synapses (paper N. 1). Gephyrin is a scaffold protein essential for stabilizing glycine and GABAA receptors at inhibitory synapses. Using recombinant intrabodies against gephyrin (scFv-gephyrin) I tested the hypothesis that this protein exerts a trans-synaptic action on GABA and glutamate release. Pair recordings from interconnected hippocampal cells in culture revealed a reduced probability of GABA release in scFv-gephyrintransfected neurons compared with controls. This effect was associated with a significant decrease in VGAT, the vesicular GABA transporter, and in neuroligin 2 (NL2), a protein that, interacting with the neurexins, ensures the cross-talk between the post- and presynaptic sites. I also found that, hampering gephyrin function produced a significant reduction in VGLUT, the vesicular glutamate transporter, an effect accompanied by a significant decrease in frequency of miniature excitatory postsynaptic currents. Over-expressing NLG2 in gephyrindeprived neurons rescued GABAergic but not glutamatergic innervation, suggesting that the observed changes in the latter were not due to a homeostatic compensatory mechanism. These results suggest a key role of gephyrin in regulating trans-synaptic signaling at both inhibitory and excitatory synapses. Several lines of evidence suggest that proteins involved in synaptic function are altered in ASDs. In particular, in a small percentage of cases, ASDs have been found to be associated with single mutations in genes encoding for cell adhesion molecules of the neuroligin-neurexin families. One of these involves the postsynaptic cell adhesion molecule neuroligin (NL) 3. In the second part of my PhD, I used transgenic mice carrying the human R451C mutation of Nlgn3, to study GABAergic and glutamatergic signaling in the hippocampus early in postnatal life (paper N. 2). I performed whole cell recordings from CA3 pyramidal neurons in hippocampal slices from NL3 R451C knock-in mice and I found an enhanced frequency of Giant Depolarizing Potentials, as compared to controls. This effect was probably dependent on an increased GABAergic drive to principal cells as demonstrated by the enhanced frequency of miniature GABAAmediated (GPSCs) postsynaptic currents, but not AMPA-mediated postsynaptic currents (EPSCs). The increase in frequency of mGPSCs suggest a presynaptic 9 type of action. This was further supported by the experiments with the fast-off GABAA receptor antagonist TPMPA that, as expected for an enhanced GABA transient in the cleft, showed a reduced blocking effect on miniature events. Although an increased number of available postsynaptic GABAA receptors, if these are not saturated by the content of a single GABA containing vesicle may account for these results, this was not the case since a similar number of receptor channels was revealed with peak-scaled non-stationary fluctuation analysis in both WT and NL3R451C knock-in mice, indicating that the observed effects were not postsynaptic in origin. Presynaptic changes in GABA release can be attributed to modifications in the probability of GABA release, in the number of release sites or in the content of GABA in single synaptic vesicles. Changes in probability of GABA release seem unlikely considering that we examined miniature events generated by the release of a single quantum. Our data do not allow distinguishing between the other two possibilities (changes in the number of release sites or in vesicle GABA content). However, in agreement with previous data from S\ufcdhof group showing an enhancement of the presynaptic GABAergic marker VGAT (but not VGlut1) in the hippocampus of NL3R451C KI mice (Tabuchi et al., 2007), it is likely that an increased GABAergic innervation may contribute to the enhancement of GABA release. In additional experiments I found that changes in frequency of miniature GABAergic events were associated with an acceleration of mGPSCs decay possibly of postsynaptic origin. The increased frequency of mEPSCs detected in adult, but not young NL3 R451C mice may represent a late form of compensatory homeostatic correction to counter the excessive GABAA-mediated inhibition. Therefore, it is reasonable to assume that alterations in the excitatory/inhibitory balance, crucial for the refinement of neuronal circuits early in postnatal development, accounts for the behavioral deficits observed in ASDs patients. Although also in the present case, a modification of gephyrin expression in R451C NL 3 knock-in mice was associated with changes in GABAergic innervations suggesting the involvement of a trans-synaptic signal, the role of NL3 mutation in this effect remains to be elucidated. Finally, I contribute in writing a review article (paper N. 3) that gives an up dated picture of alterations of GABAergic signaling present in different forms of Autism Spectrum Disorders