Recruitment and inhibitory action of hippocampal axo-axonic cells during behavior

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Temporal redistribution of inhibition over neuronal subcellular domains underlies state-dependent rhythmic change of excitability in the hippocampus

The behaviour-contingent rhythmic synchronization of neuronal activity is reported by local field potential oscillations in the theta, gamma and sharp wave-related ripple (SWR) frequency ranges. In the hippocampus, pyramidal cell assemblies representing temporal sequences are coordinated by GABAergic interneurons selectively innervating specific postsynaptic domains, and discharging phase locked to network oscillations. We compare the cellular network dynamics in the CA1 and CA3 areas recorded with or without anaesthesia. All parts of pyramidal cells, except the axon initial segment, receive GABA from multiple interneuron types, each with distinct firing dynamics. The axon initial segment is exclusively innervated by axo-axonic cells, preferentially firing after the peak of the pyramidal layer theta cycle, when pyramidal cells are least active. Axo-axonic cells are inhibited during SWRs, when many pyramidal cells fire synchronously. This dual inverse correlation demonstrates the key inhibitory role of axo-axonic cells. Parvalbumin-expressing basket cells fire phase locked to field gamma activity in both CA1 and CA3, and also strongly increase firing during SWRs, together with dendrite-innervating bistratified cells, phasing pyramidal cell discharge. Subcellular domain-specific GABAergic innervation probably developed for the coordination of multiple glutamatergic inputs on different parts of pyramidal cells through the temporally distinct activity of GABAergic interneurons, which differentially change their firing during different network states.     

Differential behavior-related activity of distinct hippocampal interneuron types during odor-associated spatial navigation

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Postnatal maturation of gamma-aminobutyric acidA and B-mediated inhibition in the CA3 hippocampal region of the rat

In the adult central nervous system, GABAergic synaptic inhibition is known to play a crucial role in preventing the spread of excitatory glutamatergic activity. This inhibition is achieved by a membrane hyperpolarization through the activation of postsynaptic γ-aminobutyric acid_A (GABA_A) and GABA_B receptors. In addition, GABA also depress transmitter release acting through presynaptic GABA_B receptors. Despite the wealth of data regarding the role of GABA in regulating the degree of synchronous activity in the adult, little is known about GABA transmission during early stages of development. In the following we report that GABA mediates most of the excitatory drive at early stages of development in the hippocampal CA3 region. Activation of GABA_A receptors induces a depolarization and excitation of immature CA3 pyramidal neurons and increases intracellular Ca²⁺ ([Ca²⁺]_i) during the first postnatal week of life. During the same developmental period, the postsynaptic GABA_B-mediated inhibition is poorly developed. In contrast, the presynaptic GABA_B-mediated inhibition is well developed at birth and plays a crucial role in modulating the postsynaptic activity by depressing transmitter release at early postnatal stages. We have also shown that GABA plays a trophic role in the neuritic outgrowth of cultured hippocampal neurons. © 1995 John Wiley & Sons, Inc.     

Ripple-selective GABAergic projection cells in the hippocampus

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The dynamics of recurrent inhibition

A heuristic model for the dynamics of recurrent inhibition, emphasizing non-linearities arising from the stoichiometry of transmitter-receptor interactions and time delays due to finite feedback pathway transmission times, is developed and analyzed. It is demonstrated that variation in model parameters may lead to the existence of multiple steady states, and the local stability of these are analyzed as well as the occurrence of switching behaviour between them. As an example of the applicability of this model, parameters are estimated for the hippocampal mossy fibre-CA3 pyramidal cell-basket cell complex. Numerically simulated responses of this system to alterations in presynaptic drive and titration of inhibitory transmitter receptors by penicillin are presented. Numerical simulations indicate the existence of multiple bifurcations between periodic solutions, as well as the existence of bifurcations to chaotic solutions, as presynaptic drive and receptor density are varied. It is hypothesized that the model offers insight into the sequences of events recorded in single CA3 pyramidal cells following the application of penicillin, a specific inhibitory receptor blocking agent.     

Density and function of central serotonin (5-HT) transporters, 5-HT1A and 5-HT2A receptors, and effects of their targeting on BTBR T+tf/J mouse social behavior

BTBR mice are potentially useful tools for autism research because their behavior parallels core social interaction impairments and restricted-repetitive behaviors. Altered regulation of central serotonin (5-HT) neurotransmission may underlie such behavioral deficits. To test this, we compared 5-HT transporter (SERT), 5-HT(1A) and 5-HT(2A) receptor densities among BTBR and C57 strains. Autoradiographic [(3) H] cyanoimipramine (1 nM) binding to SERT was 20-30% lower throughout the adult BTBR brain as compared to C57BL/10J mice. In hippocampal membrane homogenates, [(3) H] citalopram maximal binding (B(max) ) to SERT was 95 ± 13 fmol/mg protein in BTBR and 171 ± 20 fmol/mg protein in C57BL/6J mice, and the BTBR dissociation constant (K(D) ) was 2.0 ± 0.3 nM versus 1.1 ± 0.2 in C57BL/6J mice. Hippocampal 5-HT(1A) and 5-HT(2A) receptor binding was similar among strains. However, 8-OH-DPAT-stimulated [(35) S] GTPγS binding in the BTBR hippocampal CA(1) region was 28% higher, indicating elevated 5-HT(1A) capacity to activate G-proteins. In BTBR mice, the SERT blocker, fluoxetine (10 mg/kg) and the 5-HT(1A) receptor partial-agonist, buspirone (2 mg/kg) enhanced social interactions. The D(2) /5-HT(2) receptor antagonist, risperidone (0.1 mg/kg) reduced marble burying, but failed to improve sociability. Overall, altered SERT and/or 5-HT(1A) functionality in hippocampus could contribute to the relatively low sociability of BTBR mice.     

MTOR controls genesis and autophagy of GABAergic interneurons during brain development

Interneuron progenitors in the ganglionic eminence of the ventral telencephalon generate most cortical interneurons during brain development. However, the regulatory mechanism of interneuron progenitors remains poorly understood. Here, we show that MTOR (mechanistic target of rapamycin [serine/threonine kinase]) regulates proliferation and macroautophagy/autophagy of interneuron progenitors in the developing ventral telencephalon. To investigate the role of MTOR in interneuron progenitors, we conditionally deleted the Mtor gene in mouse interneuron progenitors and their progeny by using Tg(mI56i-cre,EGFP)1Kc/Dlx5/6-Cre-IRES-EGFP and Nkx2–1-Cre drivers. We found that Mtor deletion markedly reduced the number of interneurons in the cerebral cortex. However, relative positioning of cortical interneurons was normal, suggesting that disruption of progenitor self-renewal caused the decreased number of cortical interneurons in the Mtor-deleted brain. Indeed, Mtor-deleted interneuron progenitors showed abnormal proliferation and cell cycle progression. Additionally, we detected a significant activation of autophagy in Mtor-deleted brain. Our findings suggest that MTOR plays a critical role in the regulation of cortical interneuron number and autophagy in the developing brain.     

GABA Excitation during Development: the Nature of the Nurture

The lecture describes the role of GABA-mediated synaptic excitation during early ontogenesis.     

Type-1 cannabinoid receptor expression in the frog, Rana esculenta, tissues: a possible involvement in the regulation of testicular activity

Endogenous cannabinoids and type-1 cannabinoid receptor (CB1) are widely produced and distributed in the central nervous system (CNS) and peripheral nerves in mammals. In addition, the detection of endocannabinoids and corresponding receptors in non nervous peripheral tissues indicates an involvement of the system in the control of a wide range of physiological activities, including reproduction. Recently, the existence of CB1 was also observed in lower vertebrates and in urochordate suggesting that the endocannabinoid system is phylogenetically conserved. Using RT-PCR, CB1 mRNA expression profiles were characterized in a wide range of tissues of the anuran amphibian, the frog, Rana esculenta. Besides a strong expression in the CNS, CB1 was also present in testis, kidney, liver, ovary, muscle, heart, spleen, and pituitary. The CB1 expression pattern has been characterized in both testis and CNS during the annual sexual cycle. In testis, CB1 is poorly expressed during the winter stasis of the spermatogenesis rising during the breeding season and resumption period. An expression profile mismatching to that observed in testis was detected in whole-brain preparations during the sexual cycle; in particular in the diencephalon, the encephalic area mainly involved in the control of reproductive functions. Furthermore, fluctuations inside isolated encephalic areas and spinal cord were observed all over the reproductive cycle. In conclusion, CB1 receptor is expressed in R. esculenta CNS and testis. As far as the gonad it concerns, our results suggest the involvement of the endocannabinoids in the control of reproductive function.     

Cystic fibrosis transmembrane conductance regulator modulates synaptic chloride homeostasis in motoneurons of the rat spinal cord during neonatal development

Cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-regulated Cl(-) channel functional in neonatal rat spinal motoneurons. The present study investigated the developmental (P1-P8) expression of CFTR, its impact on motoneuron excitability and Cl(-) homeostasis in relation to canonical Cl(-) transporters. The Cl(-) outward transporter KCC2 gene was upregulated in females over males and increased from P1 to P8. The gene activities of the Cl(-) inward transporter NKCC1 and CFTR were positively correlated and grew between P1 and P8. P1 motoneuronal somata were immunopositive for CFTR whose expression later (P8) extended to cell processes. KCC2 immunopositivity outlined somata and cell processes at P1 and P8. Electrophysiological recording with sharp electrodes showed that the CFTR blocker glibenclamide increased motoneuron input resistance, suggesting functional CFTR in P1-P8 motoneurons. Whole cell patch-clamping of spinal motoneurons to study CFTR contribution to postnatal synaptic Cl(-) regulation indicated that glibenclamide or the selective CFTR blocker diphenylamine-2,2'-dicarboxylic acid produced a negative shift in GABA/glycine reversal potential (E(GABA/Gly) ) of spontaneously occurring synaptic events measured after block of excitatory transmission. A similar effect on E(GABA/Gly) was induced by the NKCC1 inhibitor bumetanide. A 3D reconstructed motoneuron model suggested that CFTR activity contributes to set the E(GABA/Gly) positive to the resting potential. The functional outcome of these Cl(-) mediated synaptic events depended not only on the postnatal age of the animal but also on their timing with respect to the excitatory synaptic signals. We propose that CFTR operated together with NKCC1 to produce depolarizing GABA/glycine mediated synaptic events.     

Pre-synaptic adenosine A2A receptors control cannabinoid CB1 receptor-mediated inhibition of striatal glutamatergic neurotransmission

An interaction between adenosine A(2A) receptors (A(2A) Rs) and cannabinoid CB(1) receptors (CB(1) Rs) has been consistently reported to occur in the striatum, although the precise mechanisms are not completely understood. As both receptors control striatal glutamatergic transmission, we now probed the putative interaction between pre-synaptic CB(1) R and A(2A) R in the striatum. In extracellular field potentials recordings in corticostriatal slices from Wistar rats, A(2A) R activation by CGS21680 inhibited CB(1) R-mediated effects (depression of synaptic response and increase in paired-pulse facilitation). Moreover, in superfused rat striatal nerve terminals, A(2A) R activation prevented, while A(2A) R inhibition facilitated, the CB(1) R-mediated inhibition of 4-aminopyridine-evoked glutamate release. In summary, the present study provides converging neurochemical and electrophysiological support for the occurrence of a tight control of CB(1) R function by A(2A) Rs in glutamatergic terminals of the striatum. In view of the key role of glutamate to trigger the recruitment of striatal circuits, this pre-synaptic interaction between CB(1) R and A(2A) R may be of relevance for the pathogenesis and the treatment of neuropsychiatric disorders affecting the basal ganglia.     

Regulation of ERK1/2 activity upon contact inhibition in fibroblasts

Contact inhibition is a crucial mechanism regulating proliferation in vitro and in vivo. Despite its generally accepted importance for maintaining tissue homeostasis knowledge about the underlying molecular mechanisms of contact inhibition is still scarce. Since the MAPK ERK1/2 plays a pivotal role in the control of proliferation, we investigated regulation of ERK1/2 phosphorylation which is downregulated in confluent NIH3T3 cultures. We found a decrease in upstream signaling including phosphorylation of the growth factor receptor adaptor protein ShcA and the MAPK kinase MEK1/2 in confluent compared to exponentially growing cultures whereas involvement of ERK1/2 phosphatases in ERK1/2 inactivation is unlikely. Treatment of confluent, serum-deprived cultures with PDGF-B resulted in similar phosphorylation of ERK1/2 and induction of DNA-synthesis as detected in sparse, serum-deprived cultures. In contrast, ERK1/2 phosphorylation and DNA-synthesis could not be stimulated in confluent, serum-deprived cultures exposed to EGF. Our data indicate that PDGFR- and EGFR signaling are differentially inhibited in confluent cultures of NIH3T3 cells.     

Delayed Postconditionig Initiates Additive Mechanism Necessary for Survival of Selectively Vulnerable Neurons After Transient Ischemia in Rat Brain

1. The aim of this study was to validate the role of postconditioning, used 2 days after lethal ischemia, for protection of selectively vulnerable brain neurons against delayed neuronal death.2. Eight, 10, or 15 min of transient forebrain ischemia in rat (four-vessel occlusion model) was used as initial lethal ischemia. Fluoro Jade B, the marker of neurodegeneration, and NeuN, a specific neuronal marker were used for visualization of changes 7 or 28 days after ischemia without and with delayed postconditioning.3. Our results confirm that postconditioning if used at right time and with optimal intensity can prevent process of delayed neuronal death. At least three techniques, known as preconditioners, can be used as postconditioning: short ischemia, 3-nitropropionic acid and norepinephrine. A cardinal role for the prevention of death in selectively vulnerable neurons comprises synthesis of proteins during the first 5 h after postconditioning. Ten minutes of ischemia alone is lethal for 70% of pyramidal CA1 neurons in hippocampus. Injection of inhibitor of protein synthesis (Cycloheximide), if administered simultaneously with postconditioning, suppressed beneficial effect of postconditioning and resulted in 50% of CA1 neurons succumbing to neurodegeneration. Although, when Cycloheximide was injected 5 h after postconditioning, this treatment resulted in survival of 90% of CA1 neurons.4. Though postconditioning significantly protects hippocampal CA1 neurons up to 10 min of ischemia, its efficacy at 15 min ischemia is exhausted. However, protective impact of postconditioning in less-sensitive neuronal populations (cortex and striatum) is very good after such a damaging insult like 15 min ischemia. This statement also means that up to 15 min of ischemia, postconditioning does not induce cumulation of injuries produced by the first and the second stress.     

Synaptic mechanisms of use-dependent potentiation and use-dependent inhibition of evoked burst discharges in rat hippocampal slices

The mechanisms of activity-dependent modulation of burst discharges in rat hippocampal slices have been studied. The extracellular registration of field responses (population spike, PS, and field excitatory postsynaptic potential, fEPSP) induced by repetitive electrical stimulation (1–4 Hz) of Schaffer collaterals (with 30 pulses trains separated by 5-min resting intervals) was performed in cellular and dendritic layers of CA1 area. It has been established that repetitive orthodromic stimulation exerts biphasic modulation of burst discharges in Mg²⁺-free medium: use-dependent potentiation (UDP) and use-dependent inhibition (UDI). The former was manifested as an increase in the number of PS in the burst discharge associated with a corresponding lengthening of the fEPSP. During the UDI development the number of NMDA-dependent PS in the burst was diminished despite the continuing increase in the fEPSP duration. In some cases UDI was followed by spreading depression. Both UDP and UDI were reversible. The development of UDP and UDI could be effectively suppressed either by the NMDA antagonists or by the GABAergic inhibition enhancer, diazepam. The picrotoxin (PTX)-induced burst discharges did not undergo either UDP or UDI development. However, removal of Mg²⁺ from PTX-containing solution during continuing repetitive stimulation led to the appearance of NMDA-dependent UDI. Analysis of the data obtained indicates that: (1) UDP results from a progressive decrease in GABA-mediated inhibition in the course of low-frequency (1–4 Hz) repetitive stimulation (the so-called “fatigue of synaptic inhibition”); (2) UDI is caused by excessive Ca²⁺ influx into the neurons due to overactivation of NMDA receptors. The article is published in the original.     

Activity-dependent changes of the hippocampal CA3–CA1 synapse during the acquisition of associative learning in conscious mice

Contemporary neuroscientists are paying increasing attention to subcellular, molecular and electrophysiological mechanisms underlying learning and memory processes. Recent efforts have addressed the development of transgenic mice affected at different stages of the learning process, or emulating pathological conditions involving cognition and motor-learning capabilities. However, a parallel effort is needed to develop stimulating and recording techniques suitable for use in behaving mice, in order to grasp activity-dependent neural changes taking place during the very moment of the process. These in vivo models should integrate the fragmentary information collected by different molecular and in vitro approaches. In this regard, long-term potentiation (LTP) has been proposed as the neural mechanism underlying synaptic plasticity. Moreover, N -methyl- d -aspartate (NMDA) receptors are accepted as the molecular substrate of LTP. It now seems necessary to study the relationship of both LTP and NMDA receptors with the plastic changes taking place, in selected neural structures, during actual learning. Here, we review data on the involvement of the hippocampal CA3–CA1 synapse in the acquisition of classically conditioned eyelid conditioned responses (CRs) in behaving mice. Available data show that LTP, evoked by high-frequency stimulation of Schaffer collaterals, disturbs both the acquisition of CRs and the physiological changes that occur at the CA3–CA1 synapse during learning. Moreover, the administration of NMDA-receptor antagonists is able not only to prevent LTP induction in vivo , but also to hinder the formation of both CRs and functional changes in strength of the CA3–CA1 synapse. Thus, there is experimental evidence relating activity-dependent synaptic changes taking place during actual learning with LTP mechanisms and with the role of NMDA receptors in both processes.