Scott M. Thompson

Organotypic slice cultures: a technique has come of age

Slices of CNS tissue prepared from young rodents can be maintained in culture for many weeks to months. The basic requirements are simple: a stable substratum, culture medium, sufficient oxygenation and incubation at a temperature of about 36 degrees C. Under these conditions, nerve cells continue to differentiate and to develop a tissue organization that closely resembles that observed in situ. Several alternative culturing methods have been developed recently. Slices maintained in stationary culture with the interface method are ideally suited for questions requiring a three-dimensional structure, whereas slices cultured in roller-tubes remain the method of choice for experiments that require optimal optical conditions. In this report, three typical experiments are discussed that illustrate the potential of the slice-culture technique. The first example indicates that, due to their high neuronal connectivity, slice cultures provide a very useful tool for studying the properties of synaptic transmission between monosynaptically coupled cell pairs. The other two studies show how long-term application of substances to slice cultures can be used to examine the consequences of epileptic discharges in vitro, as well as the effects of slowly acting clostridial neurotoxins on synaptic transmission.

Long-term synaptic plasticity between pairs of individual CA3 pyramidal cells in rat hippocampal slice cultures

1 Long‐term potentiation (LTP) and depression (LTD) were investigated at synapses formed by pairs of monosynaptically connected CA3 pyramidal cells in rat hippocampal slice cultures. 2 An N‐methyl‐D‐aspartate (NMDA) receptor‐mediated component of the unitary EPSP, elicited at the resting membrane potential in response to single action potentials in an individual CA3 cell, could be isolated pharmacologically. 3 Associative LTP was induced when single presynaptic action potentials were repeatedly paired with 240 ms postsynaptic depolarizing pulses that evoked five to twelve action potentials or with single postsynaptic action potentials evoked near the peak of the unitary EPSP. LTP induction was prevented by an NMDA receptor antagonist. 4 Associative LTD was induced when single presynaptic action potentials were repeatedly elicited with a certain delay after either 240 ms postsynaptic depolarizing pulses or single postsynaptic action potentials. The time window within which presynaptic activity had to occur for LTD induction was dependent on the amount of postsynaptic depolarization. LTD was induced if single pre‐ and postsynaptic action potentials occurred synchronously. 5 Homosynaptic LTD was induced by 3 Hz tetanization of the presynaptic neuron for 3 min and was blocked by an NMDA receptor antagonist. 6 Depotentiation was produced with stimulation protocols that elicit either homosynaptic or associative LTD. 7 Recurrent excitatory synapses between CA3 cells display associative potentiation and depression. The sign of the change in synaptic strength is a function of the relative timing of pre‐ and postsynaptic action potentials.

Comparison of the actions of adenosine at pre‐ and postsynaptic receptors in the rat hippocampus in vitro.

1. Intracellular microelectrode recordings were used to study the cellular location, the receptor pharmacology, and the mechanism of action of adenosine on pyramidal cells and presynaptic axonal endings in area CA3 of organotypic hippocampal slice cultures. 2. Adenosine (bath applied at 50 microM) caused a 10‐15 mV hyperpolarization of CA3 cells, as well as a 75‐100% decrease in the amplitude of excitatory and polysynaptic inhibitory postsynaptic potentials (EPSPs and IPSPs). Adenosine had no effect on the amplitude of monosynaptic IPSPs elicited in the presence of excitatory amino acid receptor antagonists, but did reduce the amplitude of isolated EPSPs, elicited after blocking GABAA receptors and reducing subsequent epileptic bursts with excitatory amino acid receptor antagonists. These data indicate that adenosine receptors are located on excitatory, but not inhibitory, presynaptic elements. 3. The A1 receptor antagonist 8‐cyclopentyl‐1,3‐dipropylxanthine (DPCPX, bath applied at 200 nM) blocked the pre‐ and postsynaptic actions of adenosine. DPCPX had no effect on the amplitude of control synaptic responses, suggesting that there is no tonic activation of adenosine receptors in hippocampal slice cultures under control conditions. The A1 receptor agonists R‐N6‐phenylisopropyladenosine (R‐PIA) mimicked all pre‐ and postsynaptic actions of adenosine. 4. Pertussis toxin pretreatment (500 ng/ml for 48 h) prevented adenosine from activating postsynaptic K+ conductance, but not from inhibiting EPSPs. In contrast, stimulation of protein kinase C with phorbol ester (phorbol 12, 13‐dibutyrate, 1 microM for 10 min) reduced the presynaptic, but not the postsynaptic, actions of adenosine. 5. Barium (bath applied at 1 mM) blocked the adenosine‐activated K+ conductance, but not the inhibition of isolated EPSPs by adenosine. 6. Adenosine at 0.03‐1 microM reduced the frequency of, or blocked, spontaneous epileptiform bursting produced by bicuculline. DPCPX (200 nM) increased the rate of spontaneous bursting, consistent with a tonic activation of adenosine receptors during hyperactivity, and led to the development of prolonged ictal‐like bursts, suggesting that the endogenous release of adenosine may contribute to the termination of epileptic bursts. 7. We conclude that adenosine acts at pre‐ and postsynaptic receptors which are pharmacologically indistinguishable. Postsynaptically, adenosine increases a barium‐sensitive K+ conductance via a pertussis toxin‐sensitive GTP‐binding protein. The presynaptic action of adenosine must, however, be mediated by some other mechanism.

Paired-pulse facilitation and depression at unitary synapses in rat hippocampus: quantal fluctuation affects subsequent release.

1. Excitatory synaptic transmission between pairs of monosynaptically coupled pyramidal cells was examined in rat hippocampal slice cultures. Action potentials were elicited in single CA3 pyramidal cells impaled with microelectrodes and unitary excitatory postsynaptic currents (EPSCs) were recorded in whole‐cell voltage‐clamped CA1 or CA3 cells. 2. The amplitude of successive unitary EPSCs in response to single action potentials varied. The amplitude of EPSCs was altered by adenosine or changes in the [Mg2+]/[CA2+] ratio. We conclude that single action potentials triggered the release of multiple quanta of glutamate. 3. When two action potentials were elicited in the presynaptic cell, the amplitude of the second EPSC was inversely related to the amplitude of the first. Paired‐pulse facilitation (PPF) was observed when the first EPSC was small, i.e. the second EPSC was larger than the first, whereas paired‐pulse depression (PPD) was observed when the first EPSC was large. 4. The number of trials displaying PPD was greater when release probability was increased, and smaller when release probability was decreased. 5. PPD was not postsynaptically mediated because it was unaffected by decreasing ionic flux with 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (CNQX) or receptor desensitization with aniracetam. 6. PPF was maximal at an interstimulus interval of 70 ms and recovered within 500 ms. Recovery from PPD occurred within 5 s. 7. We propose that multiple release sites are formed by the axon of a CA3 pyramidal cell and a single postsynaptic CA1 or CA3 cell. PPF is observed if the first action potential fails to release transmitter at most release sites. PPD is observed if the first action potential successfully triggers release at most release sites. 8. Our observations of PPF are consistent with the residual calcium hypothesis. We conclude that PPD results from a decrease in quantal content, perhaps due to short‐term depletion of readily releasable vesicles.

Presynaptic inhibition of miniature excitatory synaptic currents by baclofen and adenosine in the hippocampus

Presynaptic inhibition of neurotransmitter release is thought to be mediated by a reduction of axon terminal Ca2+ current. We have compared the actions of several known inhibitors of evoked glutamate release with the actions of the Ca2+ channel antagonist Cd2+ on action potential-independent synaptic currents recorded from CA3 neurons in hippocampal slice cultures. Baclofen and adenosine decreased the frequency of miniature excitatory postsynaptic currents (mEPSCs) without affecting the distribution of their amplitudes. Cd2+ blocked evoked synaptic transmission, but had no effect on the frequency or amplitude of either mEPSCs or inhibitory postsynaptic currents (IPSCs). Inhibition of presynaptic Ca2+ current therefore appears not to be required for the inhibition of glutamate release by adenosine and baclofen. Baclofen had no effect on the frequency of miniature IPSCs, indicating that gamma-aminobutyric acid B-type receptors exert distinct presynaptic actions at excitatory and inhibitory synapses.

Presynaptic inhibition in the hippocampus

Presynaptic receptors for virtually all transmitters have been identified throughout the nervous system. Recent studies in the hippocampus provide new insights into the mechanisms by which the activation of these receptors leads to presynaptic inhibition of transmitter release, and characterize the second messengers involved in coupling presynaptic receptors to their effectors. Presynaptic receptors also provide a tractable route via which the amount of transmitter release may be selectively regulated in therapeutically useful ways.

Mechanism of mu‐opioid receptor‐mediated presynaptic inhibition in the rat hippocampus in vitro.

1. The electrophysiological action of the mu‐opioid receptor‐preferring agonist D‐Ala2, MePhe4, Met(O)5‐ol‐enkephalin (FK 33‐824) on synaptic transmission has been studied in area CA3 of organotypic rat hippocampal slice cultures. 2. FK 33‐824 (1 microM) had no effect on the amplitude of pharmacologically isolated N‐methyl‐D‐aspartate (NMDA) or non‐NMDA receptor‐mediated EPSPs. 3. FK 33‐824 (10 nM to 10 microM) reduced the amplitude of monosynaptic inhibitory postsynaptic potentials (IPSPs) that were elicited in pyramidal cells with local stimulation after pharmacological blockade of excitatory amino acid receptors. This effect was reversible, dose‐dependent, and sensitive to naloxone and the mu‐receptor antagonist Cys2,Tyr3,Orn5,Pen7‐amide (CTOP). FK 33‐824 at 1 microM caused a mean reduction in the amplitude of the monosynaptic IPSP of 70%. 4. Neither delta‐ nor kappa‐receptor‐preferring agonists had any effect on excitatory or inhibitory synaptic potentials. 5. The disinhibitory action of FK 33‐824 was blocked by incubating the cultures with pertussis toxin (500 ng/ml for 48 h) or by stimulation of protein kinase C with phorbol 12,13‐dibutyrate (PDBu, 0.5 microM). 6. The depression of monosynaptic IPSPs by FK 33‐824 was unaffected by extracellular application of the K+ channel blockers Ba2+ or Cs+ (1 mM each). 7. FK 33‐824 produced a decrease in the frequency of miniature, action potential‐independent, spontaneous inhibitory synaptic currents (mIPSCs) recorded with whole‐cell voltage‐clamp techniques, but did not change their mean amplitude. Application of the Ca2+ channel blocker Cd2+ (100 microM) or of nominally Ca(2+)‐free solutions did not alter either the frequency and amplitude of mIPSCs or the reduction of mIPSC frequency induced by FK 33‐824. 8. The effect of FK 33‐824 on spontaneous mIPSCs was prevented by naloxone, and by incubation of cultures with pertussis toxin. 9. These results indicate that mu‐opioid receptors decrease GABA release presynaptically by a G protein‐mediated inhibition of the vesicular GABA release process, and not by changes in axon terminal K+ or Ca2+ conductances that are sensitive to extracellular Ba2+, Cs+ or Cd2+.

Either N- or P-type Calcium Channels Mediate GABA Release at Distinct Hippocampal Inhibitory Synapses

Transmitter release at most central synapses depends on multiple types of calcium channels. Identification of the channels mediating GABA release in hippocampus is complicated by the heterogeneity of interneurons. Unitary IPSPs were recorded from pairs of inhibitory and pyramidal cells in hippocampal slice cultures. The N-type channel antagonist omega-conotoxin MVIIA abolished IPSPs generated by interneurons in st. radiatum, whereas the P/Q-type antagonist omega-agatoxin IVA had no effect. In contrast, omega-agatoxin IVA abolished IPSPs generated by st. lucidum and st. oriens interneurons, but omega-conotoxin MVIIA had no effect. After unitary IPSPs were blocked by toxin, transmission could not be restored by increasing presynaptic calcium entry. The axons of the two types of interneurons terminated within distinct strata of area CA3. Thus, GABA release onto pyramidal cells, unlike glutamate release, is mediated entirely by either N- or P-type calcium channels, depending on the presynaptic cell and the postsynaptic location of the synapse.

Ca2+ or Sr2+ Partially Rescues Synaptic Transmission in Hippocampal Cultures Treated with Botulinum Toxin A and C, But Not Tetanus Toxin

Botulinum (BoNT/A–G) and tetanus toxins (TeNT) are zinc endopeptidases that cleave proteins associated with presynaptic terminals (SNAP-25, syntaxin, or VAMP/synaptobrevin) and block neurotransmitter release. Treatment of hippocampal slice cultures with BoNT/A, BoNT/C, BoNT/E, or TeNT prevented the occurrence of spontaneous or miniature EPSCs (sEPSCs or mEPSCs) as well as the [Ca2+]o-independent increase in their frequency induced by phorbol ester, 0.5 nm α-latrotoxin, or sucrose. [Ca2+]o-independent and -dependent release thus requires that the target proteins of clostridial neurotoxins be uncleaved. In contrast, significant increases in mEPSC frequency were produced in BoNT-treated, but not TeNT-treated, cultures by application of the Ca2+ionophore ionomycin in the presence of 10 mm[Ca2+]o. The frequency of sEPSCs was increased in BoNT-treated, but not TeNT-treated, cultures by increasing [Ca2+]o from 2.8 to 5–10 mm or by applying 5 mm Sr2+. Large Ca2+ and Sr2+ influxes thus can rescue release after BoNT treatment, albeit less than in control cultures. The nature of the toxin-induced modification of Ca2+-dependent release was assessed by recordings from monosynaptically coupled CA3 cell pairs. The paired-pulse ratio of unitary EPSCs evoked by two presynaptic action potentials in close succession was 0.5 in control cultures, but it was 1.4 and 1.2 in BoNT/A- or BoNT/C-treated cultures when recorded in 10 mm[Ca2+]o. Log–log plots of unitary EPSC amplitude versus [Ca2+]o were shifted toward higher [Ca2+]o in BoNT/A- or BoNT/C-treated cultures, but their slope was unchanged and the maximal EPSC amplitudes were reduced. We conclude that BoNTs reduce the Ca2+ sensitivity of the exocytotic machinery and the number of quanta released.

Presynaptic inhibition of excitatory synaptic transmission by muscarinic and metabotropic glutamate receptor activation in the hippocampus: are Ca2+ channels involved?

Activation of either muscarinic cholinergic or metabotropic glutamatergic presynaptic receptors inhibits evoked excitatory synaptic responses in the hippocampus. We have investigated two possible mechanisms underlying these actions using whole-cell recording from CA3 pyramidal cells in hippocampal slice cultures. Application of either methacholine (MCh, 10 microM) or trans-aminocyclopentane-1,3-dicarboxylic acid (t-ACPD, 10 microM) was found to reduce the frequency of miniature excitatory postsynaptic currents (mEPSCs) by roughly 50%, without changing their mean amplitude. The voltage-dependent Ca2+ channel blocker Cd2+ (100 microM), in contrast, had no effect on the mEPSC frequency. When the extracellular [K+] was increased from 2.7 to 16 mM, the mEPSC frequency increased from 1.7 to 4.9 Hz. This increase could be completely reversed by applying Cd2+, indicating that it was triggered by voltage-dependent Ca2+ influx. MCh and t-ACPD each decreased the mEPSC frequency by roughly 50% under these conditions. Because the agonists were equally effective in inhibiting spontaneous release whether voltage-dependent channels were activated or not, we conclude that presynaptic cholinergic and glutamatergic inhibition is not mediated by inhibition of presynaptic Ca2+ channels, but rather by a direct interference in the neurotransmitter release process at some point subsequent to Ca2+ influx.