Optogenetic field potential recording in cortical slices

We introduce a method that uses optogenetic stimulation to evoke field potentials in brain slices prepared from transgenic mice expressing channelrhodopsin-2-YFP. Cortical slices in a recording chamber were stimulated with a 473nm blue laser via either a laser scanning photostimulation setup or by direct guidance of a fiber optic. Field potentials evoked by either of the two optogenetic stimulation methods had stable amplitude, consistent waveform, and similar components as events evoked with a conventional stimulating electrode. The amplitude of evoked excitatory postsynaptic potentials increased with increasing laser intensity or pulse duration. We further demonstrated that optogenetic stimulation can be used for the induction and monitoring of long-term depression. We conclude that this technique allows for efficient and reliable activation of field potentials in brain slice preparation, and will be useful for studying short and long term synaptic plasticity.     

Chronic inhibition of NOS does not prevent plasticity of rat somatosensory (S1) cortex following deafferentation

Nitric oxide (NO) has been proposed as an intercellular messenger mediating postsynaptic to presynaptic information transfer in the induction of long-term potentiation. A number of studies support the possible involvement of NO in synaptic plasticity. NO may have a role in synaptogenesis and synaptic plasticity in developing rat brain and may play a fundamental part in the process of regeneration, plasticity, and retargeting of axons following injury. We examined the possible role of NO on plasticity in the rat first somatosensory cortex with [¹⁴C]2-deoxyglucose (2-DG) autoradiography in rats treated daily with -nitroarginine ( -NA) following neonatal unilateral vibrissae deafferentation. After 6 weeks of -NA treatment, the local cerebral glucose utilization (LCGU) and the spatial extent of the metabolic activation following stimulation of the spared whisker was measured. NOS catalytic activity exhibited significant inhibition throughout the treatment period. Vibrissae deafferentation produced a small but not statistically significant increase of LCGU in the vibrissa activated C3 barrel, and -NA treatment did not alter the activation of LCGU in the deafferented cortex following whisker stimulation. Additionally, -NA treatment did not alter the area of metabolic activation on either the non-deafferented side or the deafferented side. Deafferentation produced a 298% increase in the metabolic representation of the spared C3 barrel following stimulation in the saline treated animals, a 257% increase in the chronically -NA treated animals, and a 256% increase in the short-term treated animals, all with respect to the response in the non-deafferented cortex. Metabolic plasticity in the barrel cortex was not attenuated by -NA treatment. These results show that nitric oxide does not play a major role on developmental cortical plasticity induced by vibrissae deafferentation in the rat.     

The variation of caspase-3 activity in rat hippocampal slices depends on the duration of LTP maintenance

The changes in the activities of proteases after the induction of CA1 long-term synaptic plasticity in hippocampal slices from Wistar rats were investigated in the period of LTP maintenance. Electrophysiological testing was performed for 1 h after high-frequency stimulation (HFS, 100 Hz, 1 s) of Schaffer collaterals. The same testing paradigm was applied to control slices from the same animals, except for HFS. After all testing procedures, the slices were immediately frozen at −70°C and the activities of caspase-3 and calpain were measured in each slice separately. At 1 hour after HFS, caspase-3 activity decreased significantly compared to control slices. During this period, complete or partial depotentiation was observed in these slices and the percentage of recovery significantly correlated with caspase-3 activity. Moreover, decreased caspase-3 activity was observed in the slices with complete depotentiation. We suggest that HFS-induced caspase-3 inhibition may be explained by the attenuation of degenerative processes under sufficient afferent inflow. However, in the slices that were subjected to the same afferent stimulation, in which LTP maintenance was observed, caspase-3 activity was significantly higher. Thus, long-term synaptic alterations seem to be associated with recurrent caspase-3 activation. If we exclude the influence of synaptic potentiation, practically no correlation exists between caspase-3 activity and population spike amplitude during the phase of LTP maintenance. In the majority of slices, some trend to presynaptic fiber volley depression developed in parallel but independently of synaptic potentiation. In contrast, we did not reveal any calpain effects on the indices we studied. Our results suggest that different proteolytic enzymes may have specific roles under the conditions of long-term synaptic plasticity.     

Kainate receptor-mediated presynaptic inhibition converges with presynaptic inhibition mediated by Group II mGluRs and long-term depression at the hippocampal mossy fiber-CA3 synapse

Summary Kainate receptors (KARs) effect depression of glutamate release at hippocampal mossy fiber-CA3 (MF-CA3) synapses by a metabotropic action involving adenylyl cyclase (AC) inhibition, cAMP reduction, and diminished protein kinase A (PKA) activation. Using hippocampal slices, we show here that KAR activation interferes with the depression of glutamate release produced by Group II metabotropic glutamate receptor stimulation and low frequency stimulation (LFS)-induced long-term depression (LTD), also expressed through presynaptic AC/cAMP/PKA at MF-CA3 synapses. The mutual occlusion of depression mediated by presynaptic KARs, Group II mGluR and LFS-induced LTD suggests their mechanistic convergence at the MF-CA3 synapse and thus invokes KARs in synaptic plasticity manifest in LTD.     

The active zone protein CAST regulates synaptic vesicle recycling and quantal size in the mouse hippocampus

Synaptic efficacy is determined by various factors, including the quantal size, which is dependent on the amount of neurotransmitters in synaptic vesicles at the presynaptic terminal. It is essential for stable synaptic transmission that the quantal size is kept within a constant range and that synaptic efficacy during and after repetitive synaptic activation is maintained by replenishing release sites with synaptic vesicles. However, the mechanisms for these fundamental properties have still been undetermined. We found that the active zone protein CAST (cytomatrix at the active zone structural protein) played pivotal roles in both presynaptic regulation of quantal size and recycling of endocytosed synaptic vesicles. In the CA1 region of hippocampal slices of the CAST knockout mice, miniature excitatory synaptic responses were increased in size, and synaptic depression after prolonged synaptic activation was larger, which was attributable to selective impairment of synaptic vesicle trafficking via the endosome in the presynaptic terminal likely mediated by Rab6. Therefore, CAST serves as a key molecule that regulates dynamics and neurotransmitter contents of synaptic vesicles in the excitatory presynaptic terminal in the central nervous system.     

Long-term synaptic depression in the striatum: physiological and pharmacological characterization.

The effect of tetanic activation of corticostriatal glutamatergic fibers was studied in striatal slices by utilizing extracellular and intracellular recording techniques. Tetanic stimulation produced a long-term synaptic depression (LTD) (> 2 h) of both extracellularly recorded field potentials and intracellularly recorded EPSPs. LTD was not coupled with changes of intrinsic membrane properties of the recorded neurons. In some neurons, repetitive cortical activation produced a short-term posttetanic potentiation (1-3 min). Subthreshold tetanic stimulation, which under control condition did not cause LTD, induced LTD when associated with membrane depolarization. Moreover, LTD was not expressed in cells in which the conditioning tetanus was coupled with hyperpolarization of the membrane. Bath application of aminophosphonovalerate (30-50 microM), an antagonist of NMDA receptors, did not affect the amplitude of the synaptic potentials and the expression of LTD. Striatal LTD was significantly reduced by the pretreatment of the slices with 30 microM 2-amino-3-phosphonopropionic acid, an antagonist of glutamate metabotropic receptors. LTD was not blocked by bicuculline (30 microM), a GABA(A) receptor antagonist. Scopolamine (3 microM), an antagonist of muscarinic receptors, induced a slight, but significant, increase of the amplitude of LTD. Both SCH 23390 (3 microM), an antagonist of D1 dopamine (DA) receptors, and I-sulpiride (1 microM), an antagonist of D2 DA receptors, blocked LTD. LTD was also absent in slices obtained from rats in which the nigrostriatal DA system was lesioned by unilateral nigral injection of 6-hydroxydopamine. In DA-depleted slices, LTD could be restored by applying exogenous DA (30 microM) before the conditioning tetanus. In DA-depleted slices, LTD could also be restored by coadministration of SKF 38393 (3-10 microM), a D1 receptor agonist, and of LY 171555 (1-3 microM), a D2 receptor agonist. Application of a single class of DA receptor agonists failed to restore LTD. These data show that striatal LTD requires three main physiological and pharmacological conditions: (1) membrane depolarization and action potential discharge of the postsynaptic cell during the conditioning tetanus, (2) activation of glutamate metabotropic receptors, and (3) coactivation of D1 and D2 DA receptors. Striatal LTD may alter the output signals from the striatum to the other structures of the basal ganglia. This form of synaptic plasticity can influence the striatal control of motor activity.     

Chemical LTD in the CA1 field of the hippocampus from young and mature rats

Within the hippocampal formation, two forms of long-lasting synaptic plasticity, long-term potentiation (LTP) and long-term depression (LTD), can be induced which require the activation of NMDA receptors. Interestingly, it has been shown that both LTP and LTD are reduced in adult animals. Recently, a new chemical protocol has been described which elicits LTD in the CA1 field of the hippocampus. Application of 20 microM NMDA for 3 min results in a stable and long-lasting decrease in the evoked synaptic responses. We used this protocol to induce LTD in hippocampal slices from young and adult rats and show that this form of LTD is AP5-sensitive and can be blocked by the protein phosphatase inhibitor cyclosporin A in slices from adult animals. In contrast to electrical LTD (induced by prolonged low frequency stimulation), the extent of chemical LTD was not different between the young and adult rats. These findings indicate that the intracellular signal transduction cascades involved in long-lasting synaptic depression are still intact in adult animals.     

Long-term Depression of Horizontal Connections in Rat Motor Cortex

The possibility for long-term depression (LTD) of synaptic transmission in layer 11/11 I horizontal connections within motor cortex was investigated using field potentials and intracellular recordings in rat brain slices. The LTD was induced by low-frequency stimulation at 2 Hz for 10 min in sites displaced horizontally by 0.5 mm from the stimulating electrode. Response amplitude measured 25-30 min after 2 Hz stimulation ended was 79% of baseline values ( n = 13) at half maximal stimulation and 59% when 2 Hz stimulus intensity was doubled ( n = 10). In 13/15 tested cases LTD in horizontal connections was specific to the activated pathway. Intracellular recordings from six neurons confirmed synaptic character of response depression. Horizontal connections in which LTD was induced retained the capability of increasing synaptic strength. Long-term potentiation could be induced in previously depressed pathways by simultaneous theta burst stimulation of two converging horizontal inputs combined with transient local application of GABAA receptor antagonist bicuculline methiodide (mean increase: 45 ± 8%, n = 6) or by simultaneous theta burst stimulation of converging horizontal and vertical inputs (mean change: 26 5 6%, n = 5). These data demonstrate that activity-dependent mechanisms may regulate bidirectionally the effectiveness of horizontal synaptic coupling between cortical neurons, thus forming a potential mechanism for plasticity of cortical connections and the representation patterns they support.     

Glycine-induced changes in synaptic efficacy in hippocampal slices involve changes in AMPA receptors

Brief applications of high glycine concentrations to hippocampal slices have been shown to produce long-lasting changes in synaptic efficacy. In the present study, we show that glycine application transiently and reversibly increases the amplitude and prolongs the duration of synaptic potentials mediated by (NMDA) receptors. The long-lasting changes in synaptic potentials mediated by AMPA receptors are correlated with changes in the binding of [³H]α-amino-3-hydroxy-5-methylisoxazole-4-proprionic acid ([³H]AMPA) to membranes prepared from glycine-treated slices. The changes in binding properties of AMPA receptors in adult slices are due to an increase in affinity of the agonist for the receptor. Furthermore, glycine-induced increases in [³H]AMPA binding and in synaptic potentials in adult hippocampal slices are markedly reduced in the presence of low extracellular calcium or of the phospholipase inhibitor bromophenacylbromide. Finally, glycine-induced potentiation of synaptic potentials is associated with an increased potency of the glutamate receptor antagonist, 6,7-dinitroquinoxaline (DNQX), to inhibit synaptic potentials. The results indicate that glycine-induced changes in synaptic efficacy are likely triggered by the activation of NMDA receptors and expressed by changes in the properties of AMPA receptors. As similar events underly long-term potentiation (LTP), this phenomenon might provide important clues to elucidate the molecular mechanisms involved in LTP maintenance.     

Acute alcohol blocks neurosteroid modulation of synaptic transmission and long-term potentiation in the rat hippocampal slice

Effects of ethanol (22 mM) on the modulation of synaptic transmission and long-term potentiation (LTP) by the neurosteroid dehydroepiandrosterome sulfate (DHEAS; 10 μM) was examined in the in vitro rat hippocampal slice preparation. The synaptic responses were elicited by Schaffer collateral stimulation and recorded extracellularly in the somatic and dendritic regions of CA1 pyramidal neurons. LTP induction produced an increase ( 55% to 75%) in the amplitude of synaptic responses in ethanol and ethanol plus DHEAS (ethanol/DHEAS) treated slices. These increases were significantly smaller than the 130% increase observed previously in slices treated with DHEAS, but were not significantly different from the 82% increase observed in control slices. These results indicate that an ethanol/DHEAS interaction prevents the enhancement of LTP normally observed with DHEAS treatment of hippocampal slices. An ethanol/DHEAS interaction also altered DHEAS's effects on individual synaptic components of the synaptic response to Schaffer collateral stimulation. Ethanol applied before but not after DHEAS prevented DHEAS's enhancement of the NMDA receptor-mediated synaptic component. DHEAS's depression of the GABA_A receptor-mediated synaptic component was also blocked by ethanol. Ethanol or DHEAS individually had no effect on the AMPA receptor-mediated synaptic component, but application of ethanol after DHEAS resulted in a small enhancement of this synaptic component, an effect that was not observed if ethanol was applied before DHEAS. These results show that ethanol and DHEAS interact, altering DHEAS's effects on synaptic transmission and LTP in the hippocampas. Such an interaction may be involved in ethanol's actions on the CNS and raises the possibility that ethanol and DHEAS may act via a common site or pathway.     

Long-term Potentiation in the Striatum is Unmasked by Removing the Voltage-dependent Magnesium Block of NMDA Receptor Channels

We have studied the effects of tetanic stimulation of the corticostriatal pathway on the amplitude of striatal excitatory synaptic potentials. Recordings were obtained from a corticostriatal slice preparation by utilizing both extracellular and intracellular techniques. Under the control condition (1.2 mM external Mg2+), excitatory postsynaptic potentials (EPSPs) evoked by cortical stimulation were reversibly blocked by 10 microM 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), an antagonist of dl-alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) ionotropic glutamate receptors, while they were not affected by 30 - 50 microM 2-amino-5-phosphonovalerate (APV), an antagonist of N-methyl-d-aspartate (NMDA) glutamate receptors. In the presence of 1.2 mM external Mg2+, tetanic activation of cortical inputs produced long-term depression (LTD) of both extracellularly and intracellularly recorded synaptic potentials. When Mg2+ was removed from the external medium, EPSP amplitude and duration increased. In Mg2+-free medium, cortically evoked EPSPs revealed an APV-sensitive component; in this condition tetanic stimulation produced long-term potentiation (LTP) of synaptic transmission. Incubation of the slices in 30 - 50 microM APV blocked striatal LTP, while it did not affect LTD. In Mg2+-free medium, incubation of the slices in 10 microM CNQX did not block the expression of striatal LTP. Intrinsic membrane properties (membrane potential, input resistance and firing pattern) of striatal neurons were altered neither by tetanic stimuli inducing LTD and LTP, nor by removal of Mg2+ from the external medium. These findings show that repetitive activation of cortical inputs can induce long-term changes of synaptic transmission in the striatum. Under control conditions NMDA receptor channels are inactivated by the voltage-dependent Mg2+ block and repetitive cortical stimulation induces LTD which does not require activation of NMDA channels. Removal of external Mg2+ deinactivates these channels and reveals a component of the EPSP which is potentiated by repetitive activation. Since the striatum has been involved in memory and in the storage of motor skills, LTD and LTP of synaptic transmission in this structure may provide the cellular substrate for motor learning and underlie the physiopathology of some movement disorders.     

Transient decrease in F-actin may be necessary for translocation of proteins into dendritic spines

It remains poorly understood as to how newly synthesized proteins that are required to act at specific synapses are translocated into only selected subsets of potentiated dendritic spines. Here, we report that F-actin, a major component of the skeletal structure of dendritic spines, may contribute to the regulation of synaptic specificity of protein translocation. We found that the stabilization of F-actin blocked the translocation of GFP-CaMKII and inhibited the diffusion of 3-kDa dextran into spines (in 2-3 weeks cultures). Neuronal activation in hippocampal slices and cultured neurons led to an increase in the activation (decrease in the phosphorylation) of the actin depolymerization factor, cofilin, and a decrease in F-actin. Furthermore, the induction of long-term potentiation by tetanic stimulation induced local transient depolymerization of F-actin both in vivo and in hippocampal slices (8-10 weeks), and this local F-actin depolymerization was blocked by APV, a N-methyl-D-aspartate (NMDA) receptor antagonist. These results suggest that F-actin may play a role in synaptic specificity by allowing protein translocation into only potentiated spines, gated through its depolymerization, which is probably triggered by the activation of NMDA receptors.     

Low-frequency stimulation induces a new form of LTP, metabotropic glutamate (mGlu5) receptor- and PKA-dependent, in the CA1 area of the rat hippocampus

Low frequency-induced short-term synaptic plasticity was investigated in hippocampal slices with 60-electrode recording array. Remarkably, the application of low-frequency stimulation (1 Hz) for a short duration (3-5 min) resulted in the induction of a slow-onset long-term potentiation (LTP) in the immediate vicinity of the stimulated electrode. This phenomenon was observed exclusively in the CA1 subfield, neither in the CA3 area nor in the dentate gyrus. The induction of this slow-onset LTP required neither N-methyl-D-aspartate (NMDA) nor non-NMDA ionotropic receptor activation but was strongly dependent on metabotropic glutamate mGlu(5) receptor stimulation and [Ca(2+)]i increase. In addition, this form of synaptic plasticity was associated with an increase in cAMP concentration and required protein kinase A activation. Paired-pulse facilitation ratio and presynaptic fiber volley amplitude were unaffected when this LTP was triggered, suggesting the involvement of postsynaptic modifications. Although mitogen activated protein kinase pathway was stimulated after the application of low frequency, the induction and maintenance of this slow-onset LTP were not dependent on the activation of this intracellular pathway. The direct activation of adenylyl cyclase with forskolin also induced a synaptic enhancement displaying similar features. This new form of LTP could represent the mnesic engram of mild and repetitive stimulation involved in latent learning.     

Change in bi-directional plasticity at CA1 synapses in hippocampal slices taken from 6-hydroxydopamine-treated rats: the role of endogenous norepinephrine

The object of the present study is to investigate the role of endogenous adrenergic innervation in regulating bi-directional synaptic plasticity in rat hippocampal CA1 synapses. The endogenous adrenergic system was eliminated by giving subcutaneous injection of 6-hydroxydopamine (6-OHDA) to rats immediately after birth, and the animals were killed for experiments at postnatal ages of 25-35 days. In hippocampal slices taken from 6-OHDA-treated animals, theta-burst stimulation at 100 Hz failed to induce long-term potentiation (LTP) at CA1 synapses. However, the induction of long-term depression (LTD) by prolonged low frequency stimulation at 1 Hz was unaffected in slices from 6-OHDA-treated animals. Bath application of norepinephrine (NE) restored LTP to control levels and blocked LTD. This effect was mimicked by beta- but not alpha-adrenergic receptor agonists, i.e. by isoproterenol but not phenylephrine. The activators of adenylyl cyclase and protein kinase A (PKA), i.e. forskolin and 8-bromoadenosine-3', 5'-cyclic monophosphate, respectively, restored LTP in slices from 6-OHDA-treated animals. In addition, application of the D1/D5 receptor agonist, dihydrexidine, also restored LTP in slices from 6-OHDA-treated animals. These results suggest that physiologically the recruitment of catecholamine innervation may be important for induction of LTP at hippocampal CA1 synapses during tetanic stimulation, while it may not be essential for LTD induction by prolonged 1 Hz stimulation. The released NE and dopamine exert their role in modulating synaptic plasticity via activation of beta-adrenergic and D1/D5 receptors, respectively, which in turn increase the levels of cytoplasm adenosine-3',5'-cyclic monophosphate and PKA.     

Ectopic release sites lack fast vesicle recycling mechanisms, causing long-term depression of neuron-glial transmission in rat cerebellum

Classical synaptic transmission occurs at active zones within the synaptic cleft, but increasing evidence suggests that vesicle fusion can also occur outside of these zones, releasing transmitter directly into the extrasynaptic space. The role of such "ectopic" release is unclear, but in the cerebellar molecular layer it is thought to guide the processes of Bergmann glia toward synaptic terminals through activation of glial α-amino-3-hydroxyl-5-methyl-4-isoxazolepropionate (AMPA) receptors. Once surrounding the terminal, the glial process is presumed to limit spillover of neurotransmitter between synapses by rapid uptake of glutamate. We have previously reported that this route for neuron-glial transmission exhibits long-term depression following repetitive stimulation at frequencies in the 0.1-1 Hz range, in ex vivo slices from rat cerebellum. Here, we present evidence that LTD arises because ectopic sites lack the fast recycling mechanisms that operate at the active zone. Consequently, ectopic vesicles constitute an exhaustible pool that is depleted at normal synaptic firing rates and only recovers slowly. This effect is cumulative, meaning that the strength of ectopic transmission provides a read-out of the average frequency of presynaptic firing over several minutes. Glial processes are therefore likely to interact most closely with terminals that fire infrequently; conditions that may promote elimination of, rather than support for, the connection.     

Mutual inhibitory effects between dopamine and carbachol on the excitatory synaptic transmission in the rat neostriatum

The interactions between dopamine and carbachol on the excitatory synaptic transmission were studied in rat neostriatal slices using an intracellular recording method. Excitatory postsynaptic potentials (EPSPs) were evoked by cortical stimulation. Application of dopamine (DA; 0.1 μM) or carbachol (0.1 μM) produced a dramatic and reversible inhibition of the EPSP amplitude. The inhibitory effect induced by carbachol was markedly attenuated in the presence of either DA (0.1 μM) or the selective D₂ dopaminergic receptor agonist (±)-2-(N-phenylethyl-N-propyl) amino-5-hydroxytertralin (PPHT; 0.1 μM), but not by the D₁ dopaminergic receptor agonist (±)-7,8-dihydroxy-3-allyl-1-phenyl-2, 3, 4, 5-tetrahydro-1H-3-benzazepine (SKF-38393; 0.1 μM) or the D₃ dopaminergic receptor agonist R(−)-(4aS, 10bS)-3, 4, 4a, 10b-tetrahydro-4-propyl-2H, 5H-[1] benzogyrano-[4,3-b]-1, 4-oxazin-9-ol (PD-128,907; 0.1 μM). Conversely, muscarinic receptor activation with carbachol (0.1 μM) also completely abolished the DA-induced depression of the EPSP amplitude. In addition, the inhibitory effect of DA on the carbachol-induced depression of the EPSP amplitude was antagonized by sulpiride (1 μM), a selective D₂ dopaminergic receptor antagonist. However, D₁ dopaminergic receptor antagonist (±)-7-bromo-8-hydroxy-3-methyl-1-phenyl-2, 3, 4, 5-tetrahydro-3-benzazepine (SKF-83566; 1 μM) did not affect DA's inhibition. Rp-adenosine-3′,5′-cyclic monophosphothioate (Rp-cAMPS; 25 μM), a potent inhibitor of cAMP-dependent protein kinase A (PKA), alone decreased the amplitude of EPSP below baseline values and mimicked the inhibitory effect of DA on the carbachol-induced depression of the EPSP amplitude. Based on these findings, we conclude that the inhibitory effects of D₂ dopaminergic receptor and muscarinic receptor activation on the excitatory synaptic transmission in the neostriatum are non-additive and therefore are antagonistic interactions. Furthermore, the effect of muscarinic receptor stimulation will depend on the extent of D₂ dopaminergic receptor activation and the modulation of the cellular PKA-dependent messenger system seems to contribute to their interactions. © 1996 Wiley-Liss, Inc.     

The effects of cyanide on neural and synaptic function in hippocampal slices

Transverse slices from guinea pig hippocampi were exposed to micromolar concentrations of sodium cyanide while neural and synaptic function were monitored in the CA1 region. Cyanide concentrations between 10 and 200 microM rapidly depressed synaptic transmission between Schaffer collateral-commissural fibers and CA1 pyramidal cells. Analysis of input/output curves revealed that the suppression had two components, a decrease in EPSP generation and an increase in action potential threshold. Direct electrical excitability of axons was not affected. At concentrations to 500 microM, cyanide had no effect on antidromic activation of pyramidal cells. At 1000 microM, cyanide caused a moderate depression of the antidromic response in one slice while having no effect in one other. In some experiments, postsynaptic responses in the gyrus dentatus (GD), evoked by perforant path stimulation, were recorded simultaneously with CA1 responses during cyanide application. GD was found to be less sensitive to cyanide than CA1. All cyanide effects reversed rapidly and completely upon washout. These findings suggest that cyanide has a direct effect on neurons not mediated by its inhibition of metabolism.     

Modulation of the amplitude of NAD(P)H fluorescence transients after synaptic stimulation

In brain slices, excitatory synaptic stimulation results typically in transient initial decreases in NAD(P)H fluorescence, followed by longer-lasting NAD(P)H increases that overshoot pre-stimulus NAD(P)H levels before returning slowly to baseline. We concluded recently that mitochondrial metabolism (rather than NADH generation by glycolysis) was responsible for the "overshoot" phase of responses evoked in murine hippocampal slices. The present study examined factors that may influence the amplitude of the overshoot phase, without necessarily directly influencing mitochondrial or glycolytic metabolism. The amplitudes of overshoots were influenced strongly by changes in pre-stimulus NAD(P)H fluorescence levels produced by a prior electrical stimulus. In contrast, these changes in pre-stimulus redox state had little effect on the amplitude of evoked initial NAD(P)H decreases. Resting NAD(P)H fluorescence levels differed significantly across sub-regions of each slice, however, this is not due to differences in resting redox state, and the relative amplitude of NAD(P)H overshoots were not different in different slice regions. Exposure to an A1 receptor agonist (CPA) reduced the amplitude of postsynaptic potentials, and preferentially reduced the amplitude of NAD(P)H overshoots, before initial oxidizing components of biphasic transients were reduced significantly. These results suggest that amplitudes of NAD(P)H overshoots may not be good quantitative measures of the intensity of a discrete stimulus, under some conditions where the stimulus is small relative to recent activity in the slice. Comparison of flavoprotein autofluorescence with NAD(P)H levels seems useful when making quantitative comparisons of responses from different regions of slices, where optical properties and ongoing activity may be substantially different.     

Bidirectional regulation by “star forces”: Ionotropic astrocyte's optical stimulation suppresses synaptic plasticity,metabotropic one strikes back

The role of astrocytes in modulating synaptic plasticity is an important question that until recently was not addressed due to limitations of previously existing technology. In the present study, we took an advantage of optogenetics to specifically activate astrocytes in hippocampal slices in order to study effects on synaptic function. Using the AAV-based delivery strategy, we expressed the ionotropic channelrhodopsin-2 (ChR2) or the metabotropic Gq-coupled Opto-a1AR opsins specifically in hippocampal astrocytes to compare different modalities of astrocyte activation. In electrophysiological experiments, we observed a depression of basal field excitatory postsynaptic potentials (fEPSPs) in the CA1 hippocampal layer following light stimulation of astrocytic ChR2. The ChR2-mediated depression increased under simultaneous light and electrical theta-burst stimulation (TBS). Application of the type 2 purinergic receptor antagonist suramin prevented depression of basal synaptic transmission, and switched the ChR2-dependent depression into potentiation. The GABA_B receptor antagonist, phaclofen, did not prevent the depression of basal fEPSPs, but switched the ChR2-dependent depression into potentiation comparable to the values for TBS in control slices. In contrast, light stimulation of Opto-a1AR expressed in astrocytes led to an increase in basal fEPSPs, as well as a potentiation of synaptic responses to TBS significantly. A specific blocker of the Gq protein downstream target, the phospholipase C, U73122, completely prevented the effects of Opto-a1AR stimulation on basal fEPSPs or Opto + TBS responses. To understand molecular basis for the observed effects, we performed an analysis of gene expression in these slices using quantitative PCR approach. We observed a significant upregulation of “immediate-early” gene expression in hippocampal slices after light activation of Opto-a1AR-expressing astrocytes alone (cRel, Arc, Fos, JunB, and Egr1) or paired with TBS (cRel, Fos, and Egr1). Activation of ChR2-expressing hippocampal astrocytes was insufficient to affect expression of these genes in our experimental conditions. Thus, we concluded that optostimulation of astrocytes with ChR2 and Opto-a1AR optogenetic tools enables bidirectional modulation of synaptic plasticity and gene expression in hippocampus.     

Kinetics of dopamine and noradrenaline transport in synaptosomes from cerebellum, striatum and frontal cortex of normal and reeler mice.

Recent evidence indicates that the cerebellum has a dopaminergic system. In order to elucidate further the dopaminergic system in the cerebellum, we investigated the transport of dopamine (DA) in synaptosomal preparations of normal and reeler mice. For comparative purposes we also studied DA transport in synaptosomal preparations from striatum and frontal cortex and compared DA transport to noradrenaline (NA) transport. [3H]-DA transport into cerebellar synaptosomes was found to be a Na(+)-dependent, two component system--a high affinity, low capacity and a low affinity, high capacity. In striatum [3H]-DA is transported by a similar high but different low affinity component. Maximal velocities of both transport components in the striatum were higher than the corresponding ones in the cerebellum. In the frontal cortex we also observed two [3H]-DA transport components with affinities significantly lower than those in cerebellum and striatum. [3H]-NA transport into synaptosomes, prepared from the three brain regions studied, showed two transport components with similar Kt and Vmax values, except for the high affinity component in striatum whose affinity is lower. In reeler mice [3H]-DA transport was different from normal only in the cerebellum where the maximal velocity for both transport components was significantly higher (2x). In contrast, no significant difference was observed in the transport of [3H]-NA. The accumulated [3H]-DA from cerebellar slices was found to be releasable by K+ stimulation, in a Ca(++)-dependent manner, and most of the released radioactivity was in the form of [3H]-DA. These results indicate that in the cerebellum there is a low-density dopaminergic system which is distinct from the corresponding noradrenergic system.