Pair recordings reveal all-silent synaptic connections and the postsynaptic expression of long-term potentiation.

The activation of silent synapses is a proposed mechanism to account for rapid increases in synaptic efficacy such as long-term potentiation (LTP). Using simultaneous recordings from individual pre- and postsynaptic neurons in organotypic hippocampal slices, we show that two CA3 neurons can be connected entirely by silent synapses. Increasing release probability or application of cyclothiazide does not produce responses from these silent synapses. Direct measurement of NMDAR-mediated postsynaptic responses in all-silent synaptic connections before and after LTP induction show no change in failure rate, amplitude, or area. These data do not support hypotheses that synapse silent results from presynaptic factors or that LTP results from increases in presynaptic glutamate release. LTP is also associated with an increase in postsynaptic responsiveness to exogenous AMPA. We conclude that synapse silence, activation, and expression of LTP are postsynaptic.

Preparation of hippocampal brain slices.

This unit presents a procedure for the preparation of acute mammalian hippocampal slices for electrophysiological recording. Although this protocol should not be taken as the only means of making brain slices, it is a widely-used typical procedure. It is simple and straightforward. It can be used on a variety of mammalian species, though it is discussed here for use in rats.

A novel SNAP25-caveolin complex correlates with the onset of persistent synaptic potentiation.

We have identified synaptic protein complexes in intact rat hippocampal slices using the rapid chemical cross-linking reagent paraformaldehyde. Cellular proteins were rapidly cross-linked, solubilized, separated electrophoretically by SDS-PAGE, and then identified immunologically. Multiple complexes containing syntaxin, the synaptosomal-associated protein of 25 kDa (SNAP25), and vesicle-associated membrane protein (VAMP) were observed to coexist in a single hippocampal slice including a 100 kDa cross-linked protein complex that exhibited the same electrophoretic migration as a member of the previously identified SDS-resistant soluble N-ethylmaleimide-sensitive fusion attachment protein receptor "core" of the 20 S complex. A VAMP-synaptophysin complex, reported previously in vitro, was also observed in the hippocampal slices. This study links biochemical and physiological studies involving presynaptic proteins implicated in secretion and confirms that these proteins that have been studied extensively previously in the presence of detergent do form "bona fide" cellular complexes. Importantly, we have also detected additional novel protein complexes that do not correspond to complexes identified previously in vitro. After the induction of persistent synaptic potentiation, an abundant 40 kDa SNAP25-caveolin1 complex was observed. The SNAP25-caveolin1 complex was not abundant in control slices and, therefore, represents the first demonstration of a reorganization of protein complexes in intact hippocampal slices during the induction of synaptic potentiation. The interaction between caveolin1 and SNAP25 was confirmed biochemically by demonstration of the association of caveolin with recombinant-immobilized SNAP25 and by the coimmunoprecipitation of SNAP25 using caveolin-specific antisera. Caveolin1, like SNAP25, was observed to be abundant in isolated hippocampal nerve terminals (synaptosomes). Immunofluorescent studies demonstrated that both SNAP25 and caveolin1 are present in neurons and colocalize in axonal varicosities. These results suggest that a short-lasting SNAP25-caveolin interaction may be involved in the early phase of synaptic potentiation.

Presynaptic protein kinase activity supports long-term potentiation at synapses between individual hippocampal neurons.

Simultaneous microelectrode recording from two individual synaptically connected neurons enables the direct analysis of synaptic transmission and plasticity at a minimal synaptic connection. We have recorded from pairs of CA3 pyramidal neurons in organotypic hippocampal slices to examine the properties of long-term potentiation (LTP) at such minimal connections. LTP in minimal connections was found to be identical to the NMDA-dependent LTP expressed by CA3-CA1 synapses, demonstrating this system provides a good model for the study of the mechanisms of LTP expression. The LTP at minimal synaptic connections does not behave as a simple increase in transmitter release probability, because the amplitude of unitary EPSCs can increase several-fold, unlike what is observed when release probability is increased by raising extracellular calcium. Taking advantage of the relatively short axon connecting neighboring CA3 neurons, we found it feasible to introduce pharmacological agents to the interior of presynaptic terminals by injection into the presynaptic soma and have used this technique to investigate presynaptic effects on basal transmission and LTP. Presynaptic injection of nicotinamide reduced basal transmission, but LTP in these pairs was essentially normal. In contrast, presynaptic injection of H-7 significantly depressed LTP but not basal transmission, indicating a specific role of presynaptic protein kinases in LTP. These results demonstrate that pharmacological agents can be directly introduced into the presynaptic cell and that a purely presynaptic perturbation can alter this plasticity.

Muscarinic receptor activity has multiple effects on the resting membrane potentials of CA1 hippocampal interneurons.

Inhibitory interneurons appear to be an important target for the muscarinic actions of cholinergic inputs to the hippocampus. We investigated the effect of muscarinic receptor activity on the membrane potential (V(m)) and currents of rat hippocampal CA1 interneurons using whole-cell recording from visually identified CA1 interneurons. The predominant response observed was a muscarinic depolarization that was detected in interneurons from all layers of CA1. This depolarization was mediated by at least two mechanisms: a reduction in a potassium current and a mechanism that depended on extracellular sodium. Other interneurons responded to muscarinic agonists with a hyperpolarization or a biphasic response (hyperpolarization followed by depolarization). Hyperpolarizations and biphasic responses were found in all layers of CA1 but more frequently in stratum radiatum and stratum lacunosum moleculare. Muscarinic hyperpolarization was caused by the activation of a barium- and cesium-sensitive inwardly rectifying potassium channel. A small number of interneurons, primarily in or bordering the stratum pyramidale, produced slow membrane potential (0.04 Hz) oscillations. Many interneurons did not respond to muscarinic activity at all; half of these were in the stratum oriens. There was no strong correlation between any changes in V(m) response to muscarine and morphology, as determined by reconstruction of the interneurons. It was not possible to predict the morphology or the layer distribution of an interneuron based on the type of muscarinic membrane potential response it had. This lack of correlation between muscarinic function and morphology implies a greater complexity of interneuron function than has been realized previously.

Muscarinic receptor activity induces an afterdepolarization in a subpopulation of hippocampal CA1 interneurons.

Cholinergic input to the hippocampus may be involved in important behavioral functions and the pathophysiology of neurodegenerative diseases. Muscarinic receptor activity in interneurons of the hippocampus may play a role in these actions. In this study, we investigated the effects of muscarinic receptor activity on the excitability of different subtypes of interneurons in rat hippocampal CA1. Most interneurons displayed an afterhyperpolarizing potential (AHP) after depolarization by injected current or synaptic stimulation. In the presence of a muscarinic agonist, the AHP of a subset of these interneurons was replaced by an afterdepolarization (ADP), often of sufficient magnitude to evoke action potentials in the absence of further stimulation. The ADP was insensitive to cadmium and low extracellular calcium. It was blocked by low extracellular sodium but not by tetrodotoxin or low concentrations of amiloride. Muscarinic ADPs were sometimes observed in isolation but were often accompanied by depolarizing, hyperpolarizing, or biphasic changes in the membrane potential. Interneurons with muscarinic ADPs were found in all strata of CA1 and did not fall into a single morphological classification. The potential functions of the prolonged action potential output of interneurons produced by the ADP could include changes in hippocampal circuit properties and facilitation of the release of peptide cotransmitters in these interneurons.

Synaptic transmission in pair recordings from CA3 pyramidal cells in organotypic culture.

We performed simultaneous whole cell recordings from pairs of monosynaptically coupled hippocampal CA3 pyramidal neurons in organotypic slices. Stimulation of an action potential in a presynaptic cell resulted in an AMPA-receptor-mediated excitatory postsynaptic current (EPSC) in the postsynaptic cell that averaged approximately 34 pA. The average size of EPSCs varied in amplitude over a 20-fold range across different pairs. Both paired-pulse facilitation and depression were observed in the synaptic current in response to two presynaptic action potentials delivered 50 ms apart, but the average usually was dominated by depression. In addition, the amplitude of the second EPSC depended on the amplitude of the first EPSC, indicating competition between successive events for a common resource that is not restored within the 50-ms interpulse interval. Variation in the synaptic strength among pairs could arise from a variety of sources. Our data from anatomic reconstruction, 1/CV2 analysis, paired-pulse analysis, and manipulations of calcium/magnesium ratio suggest that differences in quantal size and release probability do not appear to vary sufficiently to fully account for the observed differences in amplitude. Thus it seems most likely that the variability in EPSC amplitude between pairs arises primarily from differences in the number of functional synapses. Injections of the calcium chelator bis-(o-aminophenoxy)-N, N,N',N'-tetraacetic acid into the presynaptic neuron resulted in a rapid and nearly complete block of transmission, whereas injection of the slower-acting chelator EGTA resulted in a variable and partial block. In addition to demonstrating the feasibility of manipulating the intracellular presynaptic environment by injection into the presynaptic soma, these data, and the EGTA results in particular may suggest variability in the linkage between calcium entry sites an release sites in these synapses.

Impaired synaptic plasticity in mice carrying the Huntington's disease mutation.

Cognitive impairment is an early symptom of Huntington's disease (HD). Mice engineered to carry the HD mutation in the endogenous huntingtin gene showed a significant reduction in long-term potentiation (LTP), a measure of synaptic plasticity often thought to be involved in memory. However, LTP could be induced in mutant slices by an 'enhanced' tetanic stimulus, implying that the LTP-producing mechanism is intact in mutant mice, but that their synapses are less able to reach the threshold for LTP induction. Mutant mice showed less post-tetanic potentiation than wild-type animals, and also showed decreased paired pulse facilitation, suggesting that excitatory synapses in HD mutant mice are impaired in their ability to sustain transmission during repetitive stimulation. We show that mutants, while normal in their ability to transmit at low frequencies, released significantly less glutamate during higher frequency synaptic activation. Thus, a reduced ability of Huntington synapses to respond to repetitive synaptic demand of even moderate frequency could result not only in a functional impairment of LTP induction, but could also serve as a substrate for the cognitive symptoms that comprise the early-stage pathology of HD.

Nicotinic receptor activation excites distinct subtypes of interneurons in the rat hippocampus.

We examined the function of nicotinic acetylcholine receptors (nAChRs) in interneurons of area CA1 of the rat hippocampus. CA1 interneurons could be classified into three categories based on nicotinic responses. The first class was depolarized by alpha7 nAChRs, found in all layers of CA1 and as a group, had axonal projections to all neuropil layers of CA1. The second class had both fast alpha7 and slow non-alpha7 nAChR depolarizing responses, was localized primarily to the stratum oriens, and had axonal projections to the stratum lacunosum-moleculare. The third group had no nicotinic response. This group was found in or near the stratum pyramidale and had axonal projections almost exclusively within and around this layer. Low concentrations (500 nM) of nicotine desensitized fast and slow nAChR responses. These findings demonstrate that there are distinct subsets of interneurons with regard to nicotinic receptor expression and with predictable morphological properties that suggest potential cellular actions for nicotinic receptor activation in normal CNS function and during nicotine abuse.

Basal and apical synapses of CA1 pyramidal cells employ different LTP induction mechanisms.

Nitric oxide (NO) production has been widely reported to be required for the induction of long-term potentiation (LTP) in hippocampal CA1 cells. Of the two constitutive isoforms of NO synthase, the endothelial form (eNOS) has been implicated in the induction of LTP in these cells. The distribution of eNOS within CA1 cells is not uniform, however, being present in the cell bodies and apical dendrites but absent from the basal dendrites. Using extracellular and intracellular recording techniques, we demonstrate that LTP induction in stratum radiatum synapses (onto apical dendrites) is dependent on NO production, being attenuated by pretreatment with a NOS inhibitor. LTP induced in stratum oriens synapses (onto basal dendrites) is, however, resistant to NOS inhibitors. Both forms of LTP require the activation of N-methyl-D-aspartate (NMDA) receptors because induction of LTP in both stratum radiatum and stratum oriens is blocked by AP5. Thus, it appears that synapses onto apical and basal dendrites of CA1 cells use different cellular mechanisms of LTP induction.

Excitatory actions of norepinephrine on multiple classes of hippocampal CA1 interneurons.

Norepinephrine (NE) causes an increase in the frequency of inhibitory postsynaptic potentials in CA1 pyramidal neurons in vitro. The possibility that this increase in tonic inhibition is caused by an excitatory effect on inhibitory interneurons was investigated through whole-cell recordings from pyramidal cells and both whole-cell and cell-attached patch recordings from visualized interneurons in acute slices of rat hippocampus. Adrenergic agonists caused a large increase in the frequency and amplitude of spontaneous IPSCs recorded from pyramidal cells in the presence of ionotropic glutamate receptor blockers, but they had no effect on either the frequency or the amplitude of action potential-independent miniature IPSCs recorded in tetrodotoxin. This effect was mediated primarily by an alpha adrenoceptor, although a slight beta adrenoceptor-dependent increase in IPSCs was also observed. NE caused interneurons located in all strata to depolarize and begin firing action potentials. Many of these cells had axons that ramified throughout the stratum pyramidale, suggesting that they are responsible for the IPSCs observed in pyramidal neurons. This depolarization was also mediated by an alpha adrenoceptor and was blocked by a selective alpha 1- but not a selective alpha 2-adrenoceptor antagonist. However, a slight beta adrenoceptor-dependent depolarization was detected in those interneurons that displayed time-dependent inward rectification. In the presence of a beta antagonist, NE induced an inward current that reversed near the predicted K+ equilibrium potential and was not affected by changes in intracellular Cl- concentration. In the presence of an alpha 1 antagonist, NE induced an inwardly rectifying current at potentials negative to approximately -70 mV that did not reverse (between -130 and -60 mV), characteristics similar to the hyperpolarization-activated current (lh). However, the depolarizing action of NE is attributable primarily to the alpha 1 adrenoceptor-mediated decrease in K+ conductance and not the beta adrenoceptor-dependent increase in lh. These results provide evidence that NE increases action potential-dependent IPSCs in pyramidal neurons by depolarizing surrounding inhibitory interneurons. This potent excitatory action of NE on multiple classes of hippocampal interneurons may contribute to the NE-induced decrease in the spontaneous activity of pyramidal neurons and the antiepileptic effects of NE observed in vivo.

Calcium channel involvement in GABAB receptor-mediated inhibition of GABA release in area CA1 of the rat hippocampus.

1. Experiments were performed in rat hippocampal slices to examine the nature of GABAergic inhibition of inhibitory synaptic transmission. In these experiments the effects of the gamma-aminobutyric acid-B (GABAB) receptor agonist, baclofen, and of subtype-selective calcium channel blockers were tested with the use of intracellular recordings of evoked inhibitory postsynaptic potentials (IPSPs) and whole cell recordings of spontaneous GABAergic inhibitory postsynaptic currents (IPSCs). 2. Baclofen inhibited evoked and spontaneous (action-potential-dependent) monosynaptic GABAA-mediated IPSPs and IPSCs but had no effect on the frequency of tetrodotoxin-resistant (action-potential-independent) miniature IPSCs recorded in CA1 pyramidal neurons. 3. Depolarizing GABAergic synaptic terminals by raising the extracellular potassium concentration caused an increase in action-potential-independent miniature IPSC frequency that could be inhibited by either baclofen or cadmium, a blocker of voltage-dependent calcium channels. In addition, under these depolarizing conditions, cadmium occluded the baclofen inhibition of miniature IPSCs. These data suggest that baclofen reduces only depolarization-induced, not quantal, GABA release and that it does so by decreasing presynaptic voltage-dependent calcium influx. 4. Experiments with subtype-selective calcium channel blockers demonstrate that the presynaptic action of baclofen was mediated through both omega-conotoxin-GVIA-sensitive and omega-agatoxin-IVA-sensitive, but not dihydropyridine-sensitive calcium channels.

An ADP-ribosyltransferase as a potential target for nitric oxide action in hippocampal long-term potentiation.

Recent studies of long-term potentiation (LTP) in the CA1 region of the hippocampus have demonstrated that nitric oxide (NO) may be involved in some forms of LTP and have suggested that postsynaptically generated NO is a candidate to act as a retrograde messenger. However, the molecular target(s) of NO in LTP remain to be elucidated. The present study examined whether either of two potential NO targets, a soluble guanylyl cyclase or an ADP-ribosyltransferase (ADPRT; EC 2.4.2.31) plays a role in LTP. The application of membrane-permeant analogs of cGMP did not produce any long-lasting alterations in synaptic strength. In addition, application of a cGMP-dependent protein kinase inhibitor did not prevent LTP. We found that the CA1 tissue from hippocampus possesses an ADPRT activity that is dramatically stimulated by NO and attenuated by two different inhibitors of mono-ADPRT activity, phylloquinone and nicotinamide. The extracellular application of these same inhibitors prevented LTP. Postsynaptic injection of nicotinamide failed to attenuate LTP, suggesting that the critical site of ADPRT activity resides at a nonpostsynaptic locus. These results suggest that ADP-ribosylation plays a role in LTP and are consistent with the idea that an ADPRT may be a target of NO action.

Inhibition of hippocampal heme oxygenase, nitric oxide synthase, and long-term potentiation by metalloporphyrins.

Four potent metalloporphyrin inhibitors of heme oxygenase were used to assess whether carbon monoxide production was required for induction of LTP in the CA1 region of the hippocampus. Although the metalloporphyrins produced a similar and substantial inhibition of heme oxygenase activity in hippocampal slices, only two compounds reduced the amount of LTP elicited by tetanic stimulation (chromium mesoporphyrin IX and zinc protoporphyrin IX). Both chromium mesoporphyrin IX and zinc protoporphyrin IX inhibited nitric oxide synthase in the hippocampus; tin mesoporphyrin IX and zinc deuteroporphyrin IX bis glycol neither reduced LTP induction nor inhibited NOS activity, although they did inhibit heme oxygenase. None of these metalloporphyrins reversed established LTP. Thus, together these data do not support carbon monoxide as a mediator in either LTP induction or expression/maintenance and emphasize further the nonselectivity of some metalloporphyrins.

Locally distributed synaptic potentiation in the hippocampus.

The long-lasting increase in synaptic strength known as long-term potentiation has been advanced as a potential physiological mechanism for many forms of both developmental and adult neuronal plasticity. In many models of plasticity, intercellular communication has been proposed to account for observations in which simultaneously active neurons are strengthened together. The data presented here indicate that long-term potentiation can be communicated between synapses on neighboring neurons by means of a diffusible messenger. This distributed potentiation provides a mechanism for the cooperative strengthening of proximal synapses and may underlie a variety of plastic processes in the nervous system.

Phorbol esters enhance synaptic transmission by a presynaptic, calcium-dependent mechanism in rat hippocampus.

1. The effects of phorbol esters on evoked and spontaneous excitatory neurotransmission were studied in the CA1 area in the in vitro hippocampal slice preparation of the rat. Experiments were conducted using field potential recording and whole-cell voltage clamp of CA1 pyramidal neurons. 2. Pyramidal cells dialysed during whole-cell recording with EGTA-containing electrode solutions, unable to support the induction of long-term potentiation (LTP), still showed robust phorbol ester-induced potentiation of excitatory synaptic transmission. 3. Spontaneous miniature excitatory postsynaptic currents (EPSCs), recorded in whole-cell voltage clamp in the presence of tetrodotoxin and picrotoxin, had amplitudes ranging from 4 to 40 pA and occurred at an average frequency of 0.8-5 Hz. Neither the amplitude nor the frequency of spontaneous EPSCs was altered by cadmium, dihydropyridines, or omega-conotoxin GVIA. 4. The phorbol ester 4-beta-phorbol 12,13-diacetate increased the frequency of spontaneous miniature EPSCs without changing the shape of the EPSC amplitude distribution, suggesting that phorbol esters exert their potentiating effects presynaptically. 5. Blockade of voltage-dependent calcium channels with cadmium attenuated the phorbol-induced increase in spontaneous miniature EPSCs frequency. The phorbol ester-induced increase in miniature EPSC frequency was also attenuated by dihydropyridines, but not by omega-conotoxin GVIA. 6. Unlike spontaneous synaptic currents, stimulus-evoked synaptic currents were reduced by omega-conotoxin but not by nifedipine. 7. We conclude that the phorbol ester increases spontaneous release of glutamate by modulating an L-type channel that does not participate in stimulus-evoked neurotransmitter release.