Beat H. Gähwiler

Organotypic monolayer cultures of nervous tissue

Cultivation of nervous tissue by means of the roller-tube technique yields thin organotypic cultures. Explants or slices prepared from 1- to 20-day-old rats are embedded in a plasma clot on flying coverslips and cultivated for weeks in roller-tubes. Due to the flattening of the tissue, individual nerve cells are often arranged in monolayer thickness and can, therefore, be viewed with phase-contrast microscopy. This technique is utilized to culture and co-culture nervous tissue derived from various brain regions. The degree of organotypic organization depends on the age of the animals used for culturing. Stable intracellular recordings arae obtained from nerve cells which are impaled under visual control. In view of the accessibility of individual living cells, this approach seems to be particularly well-suited for physiological and pharmacological studies on morphologically identified nerve cells.

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.

Miniature synaptic events maintain dendritic spines via AMPA receptor activation.

We investigated the influence of synaptically released glutamate on postsynaptic structure by comparing the effects of deafferentation, receptor antagonists and blockers of glutamate release in hippocampal slice cultures. CA1 pyramidal cell spine density and length decreased after transection of Schaffer collaterals and after application of AMPA receptor antagonists or botulinum toxin to unlesioned cultures. Loss of spines induced by lesion or by botulinum toxin was prevented by simultaneous AMPA application. Tetrodotoxin did not affect spine density. Synaptically released glutamate thus exerts a trophic effect on spines by acting at AMPA receptors. We conclude that AMPA receptor activation by spontaneous vesicular glutamate release is sufficient to maintain dendritic spines.

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.

Action-potential propagation gated by an axonal IA-like K+ conductance in hippocampus

Integration of membrane-potential changes is traditionally reserved for neuronal somatodendritic compartments. Axons are typically considered to transmit reliably the result of this integration, the action potential, to nerve terminals. By recording from pairs of pyramidal cells in hippocampal slice cultures, we show here that the propagation of action potentials to nerve terminals is impaired if presynaptic action potentials are preceded by brief or tonic hyperpolarization. Action-potential propagation fails only when the presynaptic action potential is triggered within the first 15–20 ms of a depolarizing step from hyperpolarized potentials; action-potential propagation failures are blocked when presynaptic cells are impaled with electrodes containing 4-aminopyridine, indicating that a fast-inactivating, A-type K+ conductance is involved. Propagation failed between some, but not all, of the postsynaptic cells contacted by a single presynaptic cell, suggesting that the presynaptic action potentials failed at axonal branch points. We conclude that the physiological activation of an IA-like potassium conductance can locally block propagation of presynaptic action potentials in axons of the central nervous system. Thus axons do not always behave as simple electrical cables: their capacity to transmit action potentials is determined by a time-dependent integration of recent membrane-potential changes.

Organotypic cultures of neural tissue

Abstract Organotypic cultures preserve the basic structural and connective organization of their tissue of origin. The high degree of neuronal differentiation, coupled with the organotypic location of neurons within a given network, permit morphological identification of types of neurons either in the living state or following histological staining. Easy access to nerve cells not only facilitates the use of standard electrophysiological techniques, but also makes these cultures amenable to patch-clamp methods. Furthermore, the successful demonstration of functional synaptic connections between co-cultured slices suggests that this technique offers a unique approach for studying synaptic transmission between remote brain areas.

Development of the hippocampus in vitro: Cell types, synapses and receptors

The postnatal development of the hippocampus and the fascia dentata have been extensively studied in a number of species. Their analysis has been facilitated by the highly characteristic structure of the hippocampus which is organized in a strictly laminated fashion. The general dynamics of hippocampal neurogenesis including dete~nation of the time of neurone origin and route of migration have been carefully investigated by [3H]thymidine autoradiography. These studies established that in rats the acquisition of pyramidal cells is completed before birth, while the majority of granule cells form during the first 3 postnatal wk.3.34s45 The present commentary describes the development of hippocampal tissue in an experimentally defined environment. For these studies, the hippocampus was removed from the brain early in its postnatal development, cut into thin slices and then cultured for periods of l-3 months. Such a procedure raises a number of general questions about intrinsic vs extrinsic developmental constraints. (1) Do the cells continue to differentiate, or does maturation depend on the presence of extrahippocampal influences, e.g. trophic factors released by alTerent fibers? (2) Do all cell types survive in culture? (3) Does synaptic reorganization occur as a response to the lack of the normal extrinsic afferents? (4) Do the cells retain normal ~hemosensiti~ty to neurotransmitters?