Properties of inhibitory and excitatory synapses between hippocampal neurons in very low density cultures

The whole cell patch clamp technique was used to examine the electrophysiological properties of embryonic hippocampal neurons maintained in a very low density (VLD) culture prejparation. The goal of these experiments was to establish the viability of the VLD culture as a model system in which to study regulation of neurotransmission at single monosynaptic connections, in the absence of polysynaptic innervation. Depolarization of neurons in the VLD culture revealed voltage-dependent sodium, calcium, and potassium currents which were blocked with, respectively, tetrodotoxin (TTX), cobalt, and tetraethylammonium and 4-aminopyridine. When pairs of neurons were simultaneously recorded, action potentials evoked in presynaptic neurons elicited either excitatory or inhibitory postsynaptic currents (EPSCs or IPSCs, respectively). The dual component “EPSCs” were due to the activation of both types-of postsynaptic, ionotropic glutamate receptors: N-methyl-D-aspartate (NMDA) and non-NMDA receptors. Evoked IPSCs were due to the activation of postsynaptic γ-aminobutyric acid (GABA) receptors. Both excitatory and inhibitory synapses exhibited short term depression in response to high frequency stimulation, although IPSCs were routinely decreased to a much greater degree than EPSCs. Spontaneous miniature EPSCs and IPSCs were found to persist in TTX, were blocked by the same pharmacological antagonists which blocked evoked responses, increased in frequency in response to hyperosmotic solution, and were unaffected by changes in extracellular calcium concentration. mIPSCS were found to occur at a significantly lower frequency than mEPSCs. These experiments indicated that neurotransmission in the VLD cultures occurs in a manner consistent with the quantal hypothesis and, therefore, the VLD culture is a good model for studying excitatory and inhibitory neurotransmission between isolated pairs of neurons. In addition, these experiments, performed under comparable physiological conditions, demonstrated that there are fundamental differences underlying neurotransmitter release between excitatory and inhibitory neurons. © 1994 Wiley-Liss, Inc.     

Postsynaptic distribution of IRSp53 in spiny excitatory and inhibitory neurons

The 53 kDa insulin receptor substrate protein (IRSp53) is highly enriched in the brain. Despite evidence that links mutations of IRSp53 with autism and other neuropsychiatric problems, the functional significance of this protein remains unclear. We used light and electron microscopic immunohistochemistry to demonstrate that IRSp53 is expressed throughout the adult rat brain. Labeling concentrated selectively in dendritic spines, where it was associated with the postsynaptic density (PSD). Surprisingly, its organization within the PSD of spiny excitatory neurons of neocortex and hippocampus differed from that within spiny inhibitory neurons of neostriatum and cerebellar cortex. The present data support previous suggestions that IRSp53 is involved in postsynaptic signaling, while hinting that its signaling role may differ in different types of neurons. J. Comp. Neurol. 522:2164–2178, 2014. © 2013 Wiley Periodicals, Inc.     

Role of microtubules and actin filaments in the movement of mitochondria in the axons and dendrites of cultured hippocampal neurons

The mitochondria in the axons and dendrites of neurons are highly motile, but the mechanism of these movements is not well understood. It has been thought that the transport of membrane-bounded organelles in axons, and perhaps also in dendrites, depends on molecular motors of the kinesin and dynein families. However, recent evidence has suggested that some organelle transport, including that of mitochondria, may proceed along actin filaments as well. The present study sought to determine the extent to which mitochondrial movements in neurons depend on microtubule-based and actin-based transport systems. The mitochondria in cultured hippocampal neurons were labeled with a fluorescent dye and the cells were treated with either nocodazole, a drug that disrupts the microtubule network or cytochalasin D or latrunculin B, drugs which disrupt the actin network. The movement of the mitochondria in the axons and dendrites of neurons after each of these drug treatments was then examined with time-lapse microscopy. Treatment with nocodazole, which depolymerizes microtubules, stopped most mitochondrial movements in both axons and dendrites. Treatment with cytochalasin D, which aggregates actin filaments, also inhibited most movements of mitochondria, but latrunculin B, which depolymerizes actin filaments, had virtually no effect. Together, these data suggest that most of the mitochondrial movements in both axons and dendrites are microtubule-based, but in each domain there may also be some movement along actin filaments.     

Some pharmacological differences between hippocampal excitatory and inhibitory synapses in transmitter release: an in vitro study.

The effects of adenosine, carbachol, and baclofen on synaptic transmission between neurons in cultured rat hippocampal explants were studied using the tight-seal whole cell clamp technique. In the culture, stimulations of neurites cause postsynaptic currents (PSCs) in nearby neurons under voltage-clamp condition. In the presence of 20 microM bicuculline, most PSCs were considered as glutamatergic excitatory postsynaptic currents (EPSCs), because they were blocked by glutamate antagonist, kynurenate at 1 mM. In the presence of 1 mM kynurenate, PSCs seemed to be inhibitory postsynaptic currents mediated by gamma-aminobutyric acid (GABA), because they were blocked by GABA antagonist, bicuculline at 20 microM. Adenosine at 100 microM and carbachol at 10 microM suppressed these EPSCs to about 35% of control. However, adenosine and carbachol at the same concentration did not suppress the IPSCs. Baclofen at 10 microM suppressed both EPSCs and IPSCs significantly (EPSCs: to about 40% of control, IPSCs: to about 30% of control). In contrast, membrane currents elicited by ionophoretically applied glutamate and GABA were not suppressed by 100 microM adenosine, 10 microM carbachol, and 10 microM baclofen. From these results, it is suggested that the pharmacological sensitivities of transmitter release from presynaptic terminals are different between glutamatergic excitatory synapses and GABAergic inhibitory synapses in hippocampal cultures.     

Differential presynaptic modulation of excitatory and inhibitory autaptic currents in cultured hippocampal neurons

Short-term synaptic plasticity has an important role in higher cortical function. Hyperpolarization may effect a form of short-term plasticity by promoting recovery from sodium channel inactivation or by activating axonal A-type potassium channels. To determine whether one or both processes occur, we examined the effect of hyperpolarizing prepulses on autaptic currents in cultured postnatal rat hippocampal neurons. As expected of enhanced recovery from sodium channel inactivation, hyperpolarizing prepulses reversibly increased fast excitatory autaptic currents (eacs) mediated by alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs), slow eacs mediated by N-methyl-D-aspartate receptors (NMDARs), and inhibitory autaptic currents (iacs) mediated by gamma-aminobutyric acidA receptors (GABAARs). Hyperpolarizing prepulses augmented nearly all fast and slow eacs but only half of the iacs. This change occurred without a change in autaptic current kinetics. Of note, hyperpolarizing prepulses did not significantly reduce autaptic currents in any neuron studied. The rapidly dissociating competitive antagonists kynurenate and L-2-amino-5-phosphonovaleric acid (LAPV) inhibited fast and slow eacs, respectively, to the same extent with and without a hyperpolarizing prepulse. In addition, hyperpolarizing prepulses revealed a slow eac even after the slow eac evoked without a prepulse was completely blocked by the open channel blocker, (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine hydrogen maleate (MK-801). Finally, hyperpolarizing prepulses did not alter currents evoked by exogenous applications of glutamate and GABA. These findings suggest that hyperpolarizing prepulses preferentially enhance eacs over iacs, and that they do so, in part, by overcoming conduction block or by activating silent synapses.     

Differences in multiple forms of short-term plasticity between excitatory and inhibitory hippocampal neurons in culture

Synaptic transmission is highly dynamic, especially during periods of repetitive activity. This short-term synaptic plasticity, elicited by either pairs or short trains of action potentials at moderate frequencies (1-10 Hz), may give rise to either depression or facilitation of synaptic transmission. We analyzed these processes in isolated, synaptically coupled pairs of inhibitory or excitatory neurons grown in low-density cultures of hippocampal neurons. Most inhibitory and excitatory synapses in these cultures displayed paired pulse depression, although the responses of excitatory synapses were more variable and occasionally facilitation was seen. With tetanic stimuli, inhibitory synapses showed depression, but excitatory synapses showed a much richer repertoire of behaviors, including depression and facilitation. While many inhibitory synapses showed posttetanic depression following short trains of action potentials, excitatory synapses instead showed posttetanic facilitation. This facilitation is accompanied by an increase in paired pulse ratio, suggesting that it is the result of presynaptic mechanisms. Finally, excitatory synapses often displayed paired pulse and tetanic facilitation of asynchronous release, a process not seen in inhibitory synapses in these cultures. These similarities and differences in short-term plasticity exhibited by inhibitory and excitatory cells are likely to be critical for information processing and the control of neuronal excitability, under both normal and pathological conditions, such as epilepsy.     

Formin' bridges between microtubules and actin filaments in axonal growth cones

<正>It is well established that guidance of axons during neuronal development is regulated by a variety of extracellular signals,governing cytoskeletal dynamics in growth cones.The actin and microtubule(MT)cytoskeleton have both been shown to play important roles.However,a growing body of work suggests that a critical issue is the proper coordination of changes within these two major cytoskeletal systems(reviewed in Cammara-     

Miniature excitatory synaptic currents in cultured hippocampal neurons.

We performed patch clamp recordings in the whole cell mode from cultured embryonic mouse hippocampal neurons. In bathing solutions containing tetrodotoxin (TTX), the cells showed spontaneous inward currents (SICs) ranging in size from 1 to 100 pA. Several observations indicated that the SICs were miniature excitatory synaptic currents mediated primarily by non-NMDA (N-methyl-D-aspartate) excitatory amino acid receptors: the rising phase of SICs was fast (1 ms to half amplitude at room temperature) and smooth, suggesting unitary events. The SICs were blocked by the broad-spectrum glutamate receptor antagonist gamma-D-glutamylglycine (DGG), but not by the selective NMDA-receptor antagonist D-2-amino-5-phosphonovaleric acid (5-APV). SICs were also blocked by desensitizing concentrations of quisqualate. Incubating cells in tetanus toxin, which blocks exocytotic transmitter release, eliminated SICs. The presence of SICs was consistent with the morphological arrangement of glutamatergic innervation in the cell cultures demonstrated immunohistochemically. Spontaneous outward currents (SOCs) were blocked by bicuculline and presumed to be mediated by GABAA receptors. This is consistent with immunohistochemical demonstration of GABAergic synapses. SIC frequency was increased in a calcium dependent manner by bathing the cells in a solution high in K+, and application of the dihydropyridine L-type calcium channel agonist BAY K 8644 increased the frequency of SICs. Increases in SIC frequency produced by high K+ solutions were reversed by Cd2+ and omega-conotoxin GVIA, but not by the selective L-type channel antagonist nimodipine. This suggested that presynaptic L-type channels were in a gating mode that was not blocked by nimodipine, and/or that another class of calcium channel makes a dominant contribution to excitatory transmitter release.     

Differential depression at excitatory and inhibitory synapses in visual cortex

The function of cortical circuits depends critically on the balance between excitation and inhibition. This balance reflects not only the relative numbers of excitatory and inhibitory synapses but also their relative strengths. Recent studies of excitatory synapses in visual and somatosensory cortices have emphasized that synaptic strength is not a fixed quantity but is a dynamic variable that reflects recent presynaptic activity. Here, we compare the dynamics of synaptic transmission at excitatory and inhibitory synapses onto visual cortical pyramidal neurons. We find that inhibitory synapses show less overall depression than excitatory synapses and that the kinetics of recovery from depression also differ between the two classes of synapse. When excitatory and inhibitory synapses are stimulated concurrently, this differential depression produces a time- and frequency-dependent shift in the reversal potential of the composite postsynaptic current. These results indicate that the balance between excitation and inhibition can change dynamically as a function of activity.     

Separate inhibitory and excitatory components underlying receptive field organization in superficial medullary dorsal horn neurons

Extracellular recording has shown that dorsal horn neurons can have an inhibitory surround outside their excitatory receptive field, but cannot reveal inhibitory inputs within the excitatory field, or show the underlying excitatory and inhibitory synaptic inputs that determine net output. To study the underlying components of receptive field organization, in vivo patch-clamp recording was used to compare the size and distribution of subthreshold, suprathreshold, and inhibitory fields, in neurons in the mouse superficial medullary dorsal horn that were characterized by their responses to noxious and innocuous mechanical facial stimulation. Subthreshold excitatory fields typically extended some distance beyond the borders of the suprathreshold field, and also commonly exhibited broader stimulus selectivity, in that the majority of nociceptive-specific neurons exhibited subthreshold responses to brush. Separate voltage-clamp recording of excitatory and inhibitory inputs using different holding potentials revealed that inhibition could be evoked from both within and outside the excitatory field. In nociceptive neurons, inhibition tended to be maximal at the excitatory receptive field center, and was usually greater for pinch than brush, although the selectivity for pinch versus brush was not as great as with excitatory responses. Based on current data on dorsal horn organization, we propose that the localized peak of inhibition at the excitatory field center could be mediated by local interneurons, while the more widespread surrounding inhibition may depend on supraspinal circuitry.     

Transport of neurofilaments in growing axons requires microtubules but not actin filaments

Neurofilament (NF) polymers are conveyed from cell body to axon tip by slow axonal transport, and disruption of this process is implicated in several neuronal pathologies. This movement occurs in both anterograde and retrograde directions and is characterized by relatively rapid but brief movements of neurofilaments, interrupted by prolonged pauses. The present studies combine pharmacologic treatments that target actin filaments or microtubules with imaging of NF polymer transport in living axons to examine the dependence of neurofilament transport on these cytoskeletal systems. The heavy NF subunit tagged with green fluorescent protein was expressed in cultured sympathetic neurons to visualize NF transport. Depletion of axonal actin filaments by treatment with 5 microM latrunculin for 6 hr had no detectable effect on directionality or transport rate of NFs, but frequency of movement events was reduced from 1/3.1 min of imaging time to 1/4.9 min. Depolymerization of axonal microtubules using either 5 microM vinblastine for 3 hr or 5 microg/ml nocodazole for 4-6 hr profoundly suppressed neurofilament transport. In 92% of treated neurons, NF transport was undetected. These observations indicate that actin filaments are not required for neurofilament transport, although they may have subtle effects on neurofilament movements. In contrast, axonal transport of NFs requires microtubules, suggesting that anterograde and retrograde NF transport is powered by microtubule-based motors.     

Direction selectivity of excitatory and inhibitory neurons in ferret visual cortex.

Direction selectivity is a characteristic feature of neurons in the visual cortex of higher mammals. Excitatory and inhibitory cortical neurons receive different patterns of synaptic connections resulting in different receptive field properties. We have analyzed the direction tuning of excitatory and inhibitory neurons of ferret visual cortex using single unit recordings. Direction tuning was constant among neurons in a vertical column. The majority (> 80%) of excitatory (regular spiking) neurons were direction tuned or direction biased. Fast spiking (inhibitory) neurons were orientation, but only weakly or not direction tuned. This indicates that excitatory and inhibitory neurons have different functions in visual processing and their different integration in thalamocortical and intracortical circuits results in a diversification of receptive field properties.     

Trophic influences of excitatory and inhibitory synapses on neurones in the auditory brain stem.

The effects of a reduction during development of excitatory and inhibitory synaptic input on CNS neurones were studied in the lateral superior olivary nucleus (LSO) of the ferret following neonatal, unilateral cochlear removal. LSO neurones normally receive excitatory input from the ipsilateral ear and inhibitory input from the contralateral ear. After cochlear removal, the ipsilateral LSO was smaller and contained fewer neurones than either the contralateral or the normal LSO. No difference was found between the volume or number of neurones in the latter nuclei. Remaining neurones in the LSO ipsilateral to the removal were smaller than those in the contralateral LSO of the same ferrets. These data show that activity in excitatory pre-synaptic terminals can be sufficient for post-synaptic target maintenance, but that activity in inhibitory terminals alone is not.     

Network stability through homeostatic scaling of excitatory and inhibitory synapses following inactivity in CA3 of rat organotypic hippocampal slice cultures

Homeostatic plasticity is a phenomenon whereby synaptic strength is scaled in the context of the activity that the network receives. Here, we have analysed excitatory and inhibitory synapses in a model of homeostatic plasticity where rat organotypic hippocampal slice cultures were deprived of excitatory synaptic input by the NMDA and AMPA/KA glutamate receptor antagonists, AP5 and CNQX. We show that chronic excitatory synapse deprivation generates an excitable CA3 network where enhanced amplitude and frequency of spontaneous excitatory post-synaptic potentials were associated with increased glutamate receptor subunit expression and increased number and size of synapsin 1 and VGLUT1 positive puncta. Intact spontaneous inhibitory post-synaptic potentials coincided with persistent expression of the GABA-A receptor alpha subunit and GAD65 and an enhancement of parvalbumin-positive puncta. In this model of homeostatic plasticity, scaling up of synaptic excitation and maintenance of fast synaptic inhibition promote an excitable, but stable, CA3 network.     

Kalirin‐7, an important component of excitatory synapses,is regulated by estradiol in hippocampal neurons

Estradiol enhances the formation of dendritic spines and excitatory synapses in hippocampal neurons in vitro and in vivo, but the underlying mechanisms are not fully understood. Kalirin‐7 (Kal7), the major isoform of Kalirin in the adult hippocampus, is a Rho GDP/GTP exchange factor localized to postsynaptic densities. In the hippocampus, both Kal7 and estrogen receptor α (ERα) are highly expressed in a subset of interneurons. Over‐expression of Kal7 caused an increase in spine density and size in hippocampal neurons. To determine whether Kalirin might play a role in the effects of estradiol on spine formation, Kal7 expression was examined in the hippocampus of ovariectomized rats. Estradiol replacement increased Kal7 staining in both CA1 pyramidal neurons and interneurons in ovariectomized rats. Estradiol treatment of cultured hippocampal neurons increased Kal7 levels at the postsynaptic side of excitatory synapses and increased the number of excitatory synapses along the dendrites of pyramidal neurons. These increases were mediated via ERα because a selective ERα agonist, but not a selective ERβ agonist, caused a similar increase in both Kal7 levels and excitatory synapse number in cultured hippocampal neurons. When Kal7 expression was reduced using a Kal7‐specific shRNA, the density of excitatory synapses was reduced and estradiol was no longer able to increase synapse formation. Expression of exogenous Kal7 in hippocampal interneurons resulted in decreased levels of GAD65 staining. Inhibition of GABAergic transmission with bicuculline produced a robust increase in Kal7 expression. These studies suggest Kal7 plays a key role in the mechanisms of estradiol‐mediated synaptic plasticity. © 2010 Wiley‐Liss, Inc.     

Activity-dependent formation of perforated synapses in cultured hippocampal neurons

The study investigated the formation of perforated synapses in rat hippocampal cell cultures. Perforated synapses are defined by their discontinuous postsynaptic densities (PSDs) and are believed to occur in parallel with changes in synaptic activity and possibly also synaptic efficacy. Several in vivo studies have demonstrated an increase in the frequency of perforated synapses induced by development and environmental stimulation as well as long-term potentiation (LTP). Also in in vitro brain slices, LTP was associated with an elevated number of perforated spine synapses. Our study demonstrated for the first time that the formation of perforated synapses can be induced by a short-term increase in spontaneous neural activity in a hippocampal cell culture model. Stimulation with the GABAA-antagonist picrotoxin (PTX) induced a significant increase in the percentage of perforated synapses. This strong increase was blocked when APV was added together with PTX, indicating that the formation of perforated synapses depended on the activation of NMDA receptors. We also showed that inhibition of the tissue type plasminogen activator (tPA-stop/PAI-1) significantly interfered with the activity-induced increase in perforated synapses. This implies that the proteolytic activities of tPA might be involved in steps which are downstream from the NMDA receptor-mediated synaptic plasticity leading to structural changes at synaptic contacts. In contrast, even long-term inhibition of electrical network activity by tetrodotoxin had no effect on the number of perforated synapses, but almost completely abolished the formation of spine synapses. These results indicate that a short-term increase in neural activity via NMDA receptors and a proteolytic cascade involving tPA lead to the formation of perforated synapses.     

Development of gamma-aminobutyric acidergic synapses in cultured hippocampal neurons

The formation and maturation of gamma-aminobutyric acid (GABA)-ergic synapses was studied in cultured hippocampal pyramidal neurons by both performing immunocytochemistry for GABAergic markers and recording miniature inhibitory postsynaptic currents (mIPSCs). Nascent GABAergic synapses appeared between 3 and 8 days in vitro (DIV), with GABAA receptor subunit clusters appearing first, followed by GAD-65 puncta, then functional synapses. The number of GABAergic synapses increased from 7 to 14 DIV, with a corresponding increase in frequency of mIPSCs. Moreover, these new GABAergic synapses formed on neuronal processes farther from the soma, contributing to decreased mIPSC amplitude and slowed mIPSC 19-90% rise time. The mIPSC decay quickened from 7 to 14 DIV, with a parallel change in the distribution of the alpha5 subunit from diffuse expression at 7 DIV to clustered expression at 14 DIV. These alpha5 clusters were mostly extrasynaptic. The alpha1 subunit was expressed as clusters in none of the neurons at 7 DIV, in 20% at 14 DIV, and in 80% at 21 DIV. Most of these alpha1 clusters were expressed at GABAergic synapses. In addition, puncta of GABA transporter 1 (GAT-1) were localized to GABAergic synapses at 14 DIV but were not expressed at 7 DIV. These studies demonstrate that mIPSCs appear after pre- and postsynaptic elements are in place. Furthermore, the process of maturation of GABAergic synapses involves increased synapse formation at distal processes, expression of new GABAA receptor subunits, and GAT-1 expression at synapses; these changes are reflected in altered frequency, kinetics, and drug sensitivity of mIPSCs.