Patch pipettes are more useful than initially thought: simultaneous pre- and postsynaptic recording from mammalian CNS synapses in vitro and in vivo
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KeywordsPatch pipette CNS synapses Thalamocortical pathway Calyx of Held
Patch pipettes were designed, initially, for the purpose of recording ionic currents through individual channels in cell membranes using a single pipette placed on a muscle membrane . Here, we could watch a single biological molecule as it changed its structure by opening and closing a transmembrane pore. For example, at acetylcholine (ACh) concentrations of about 1 μM or higher, the same molecule switches repeatedly between an open and several closed state(s) . The formation of a GΩ-seal between pipette tip and membrane widened the field of ion channel research. Currents were recorded with submillisecond resolution to resolve short opening and closing events . Combined with genetic manipulation of ion channels expressed heterologously in Xenopus laevis oocytes, this technique facilitated the first structure–function assignments of ion channels. Single amino acids in putative pore-forming domains could be exchanged, and the subsequent effect on ion flows could be directly measured . Finally, current recordings could be made from cell compartments such as the dendrite and terminal of a nerve cell.
“Patch pipettes are, however, more useful than initially thought.” (Fred Sigworth)
The whole-cell recording configuration also opened up a new field of research into the synapses of the mammalian central nervous system (CNS). In conjunction, the development of novel in vitro brain slice preparations and new visualization techniques in slices [12, 36] allowed us to characterize the properties of synapses in anatomically identified CNS pathways. Most useful were simultaneous whole-cell recordings made from cell pairs under visual control in combination with post hoc reconstructions of dendrites and axons of the two cells . I will review the results we obtained by simultaneous pair recordings from pre- and postsynaptic CNS neurons and anatomical reconstructions. Each of the synapses we studied has a different function, and each is favorable to examine one particular aspect of synaptic function.
One question relating to the properties of synapses in a neuronal network is how they might contribute to the magnitude and variability of AP responses observed in sensory systems. Unit recordings from single cells in sensory cortices indicate large differences both in the strength (efficacy) and trial-to-trial variability of suprathreshold responses. The contribution of synapses to this variability in particular is, so far, not well understood. By comparing three different synapses that are components of sensory pathways, we illustrate how one might be able to identify their contribution to the reliability of sensory representations. The rationale of this approach is to compare in vivo the magnitude and variability of response to sensory stimuli at different stages of a pathway. Concomitantly, a particular synapse between two stages is studied in vitro to delineate factors that determine synaptic efficacy and reliability.
We first studied in vitro a relay synapse in the acoustic pathway. Cell pairs form an axosomatic synapse where the nerve terminal (the calyx of Held) is in direct contact with the postsynaptic cell soma. Here, the tips of the two recording pipettes are separated by less than 10 μm, and both can be viewed and manipulated under visual control. Secondly, also in vitro, we studied a local circuit synapse in the neocortex where the tips of the two pipettes that are used to record from pre- and postsynaptic cells in the neocortex are separated by ≈ 200–1,000 μm. Here, the field of view has to be changed for visualization of pipette tips when attaching one of the two pipettes to a soma of a pair. This necessitated the development of new, movable microscope stages for visualization and micromanipulation of pipette tip positions under visual control. In contrast, recordings to the calyx preparation are restricted to the two somata of pre- and postsynaptic cells, meaning that the synaptic contacts are located electrically remote from the recording pipette tips. Thirdly, more recently, we characterized in vivo a synapse in the thalamocortical (tc) pathway. This pathway “couples” the afferent somatosensory system to the neocortex. Here, the tips of the pair of patch pipettes are separated by several millimeters and (so far) cannot be visualized during the experiment. Finding a monosynaptic connection between the ventral posterior nucleus of the thalamus (VPM) and cortex relies upon topographically aligning the recording pipettes in the two brain regions and on a high density of individual tc axons and postsynaptic cortical dendrites.
Relay synapse between two auditory nuclei in the brain stem
The major determinant of reliable and phase-locked synaptic transmission is the time course of release probability, which in turn is governed by the size and time course of the local [Ca2+] transient at vesicular release sites, operationally defined as the Ca2+ sensor. As the spatial relationship between the vesicle release sites, the Ca2+ entry sites, and the endogenous Ca2+ buffers is uncertain, the [Ca2+]i dynamics at the site of the Ca2+ sensor were also unknown. We established recording conditions  that allowed us, eventually , to determine the time course and amplitude of the local [Ca2+]i transient during an AP in the close vicinity of a vesicular release site (Fig. 2e). We estimated the size and the time course of the local [Ca2+]i transient initially by back-calculating of the [Ca2+]i transient during an AP using the experimentally determined kinetic parameters of the putative Ca2+ sensor that triggers release (Fig. 2c; [3, see also 34]). However, it remained unclear whether very short transient [Ca2+]i elevations were sufficient to trigger release similar to that observed during an AP. We mimicked the [Ca2+]i transient during an AP by shaping the time course and amplitude of the [Ca2+]i transient evoked by an intracellular UV photolyzable Ca2+ cage via a UV flash. Shaping the transient was achieved by including a small amount of nonphotolyzable Ca2+ buffer in the presynaptic pipette solution. The EPSC in response to a short [Ca2+]i transient is comparable in size and time course to that evoked by a single AP when the duration of the [Ca2+]i transient is appropriately shaped (Fig. 2d; ). This measurement is in reasonable agreement with the initial back-calculated time course (Fig. 2c).
The local [Ca2+] transient at the release site of AZs (Fig. 2e) evoked by an AP lasts for < 500 μs with a peak of 15–20 μM at the putative Ca2+ sensor that acts as a Ca2+ concentration follower. The duration of high release probability is very short due to the short Ca2+ current. The high sensitivity of release to the time course of the [Ca2+]i transient may likely be a mechanism by which the waveform of the presynaptic AP modulates the release probability (p r) and thereby synaptic efficacy.
Local circuit synapse between two layers of neocortex
Thus, a single L4–L2/3 connection, that is part of the pathway which mediates tactile stimulus representation in the supragranular layer of the somatosensory cortex, is “weak” but “reliable”. This finding suggests that the sparse AP representation and the high variability in response to tactile stimuli in L2/3 are not dominated by the synaptic efficacy of individual L4–L2/3 connections. Rather, it reflects a network property of the ensemble of L4 cells connecting to the ensemble of L2/3 cells such as the low synchrony of L4 cell responses and their sparseness to simple stimuli like a single whisker deflection.
Two limiting types of excitatory CNS synapses
In a simplified view, derived from the neuromuscular junction , the synaptic efficacy (or strength of a synapse) is described by the size of the PSP evoked by a single presynaptic AP, as PSP = n f·q·p r. Here, n f is the number of vesicle release sites or AZs, q is the quantal size, and p r is the average release probability at each site. For both synapses that we examined, the estimates of n f were close to the estimates of n a, suggesting that the single vesicle hypothesis  applies to these mammalian CNS synapses. Synaptic efficacy in the CNS thus can formally be described by the above relation, and it is useful to compare the efficacy of the two types of synapses and the factors determining it. Eventually, one might relate them to specific functions in their respective networks.
The intracortical L4 spiny stellate–L2/3 pyramid synapse is, possibly, another prototypical CNS synapse. This synapse is part of the L4–L2/3 signaling pathway that transmits excitation from the recipient granular layer to the supragranular layers of cortex. The function of the pathway is to transmit excitation from the deflection of a single or multiple whisker(s) and allow integration of excitation in L2/3 cells by their lateral axon collaterals in supra- and infragranular layers. Eventually, the integrated excitation generated by deflection of whiskers is conveyed, via long-range projections from L2/3, to other cortical areas (e.g., motor cortex, association cortex, etc.). The synaptic efficacy of individual L4–L2/3 connections is low, with an average unitary EPSP of ∼ 0.6 mV. Synapses are weak because of a small number of contacts (≈ 5 AZs) established between L4 and L2/3 neurons and a small quantal size of < 200 μV, but each connection transmits reliably.
How does synaptic efficacy and variability contribute to the sensory evoked response of a population of cells and its trial-to-trial variability in a CNS pathway? In the auditory pathway, the click response of MNTB neurons are phase-locked APs. There is little difference between AVCN and MNTB cell responses, and the sensory responses in both nuclei are effective and reliable . In the afferent somatosensory system, the response to a tactile stimulus in the supragranular layer L2/3 is mediated by the L4-to-L2/3 pathway. Here, the response is sparse (< 1 AP) and variable for an individual L2/3 pyramid and for the population of L2/3 pyramids , (de Kock et al., submitted). For comparing the possible synapse-related factors that contribute to the very different AP responses to a sensory stimulus in the MNTB and in L2/3, one has to take into account the number of presynaptic neurons that converge on a postsynaptic neuron. In the AVCN–MNTB pathway, convergence is 1:1, the lower limit value (Fig. 5a). Typically, ∼ 600 synaptic contacts are established by a single connection . Of these, about 200 AZs are active upon a sensory stimulus. In the cortex L4–L2/3 pathway, the number of contacts in an individual connection is low, ~4.5 contacts on average . However, convergence between L4 spiny cells and L2/3 pyramidal cells is high, about 300–400:1 . Thus, each pyramid in L2/3 is targeted by ∼ 1,500 AZs from L4, each of which is transmitting reliably following an AP (at least in vitro). Between 10 and 40% of L4 cells are generating APs upon a tactile stimulus  (de Kock et al., submitted), controlling release from ∼ 120–480 AZs of L4 cell terminals contacting a single L2/3 cell. With this estimate of active AZs, the evoked responses in L2/3 could be tens of mV and reliable (≥ 1 AP/stimulus). Here, however, population synchrony of APs in L4 and thus of release from L4 spiny stellate terminals is low when compared with AVCN in the brainstem pathway, where all AZs are under the control of one AP. One may therefore infer that one main source of sparse single cell responses (< 1 AP) and their higher variability in cortical L2/3 is the weaker synchrony of APs in the population of presynaptic L4 cells (Fig. 5b). Experimentally, this difference in synchrony of release in the two pathways is demonstrated by the short latency between EPSP onset and AP onset in MNTB neurons (< 0.5 ms, Fig. 2a) and the more than tenfold longer latency between EPSP onset and AP onset in L2/3 pyramids .
Thus, the functional differentiation of the calyx–principal neuron synapse within the AVCN–MNTB pathway seems to underlie a well-defined function of the acoustic system, sound localization. The contribution of the spiny stellate–pyramid cell synapse to cortical representation of tactile stimuli is less clear. One may speculate that the reliable transmission of individual connections in the L4–L2/3 pathway may be a requirement for the detection of stimulus-evoked synchronous input into the cortex, for example, for the detection of the direction of whisker deflections or of their temporal order. Such population synchrony in L4 would evoke reliably larger AP responses in L2/3. This view is supported by the experiments described in the next section, where the determination of the sensory response by the synchrony of converging inputs is illustrated for the tc projection.
In vivo pair recording from a projection synapse connecting thalamus and cortex
One drawback inherent to the acute brain slice is the fact that in vitro, a large portion of (presynaptic) axonal arbors is removed. This prevents measurements with simultaneous pair recordings that are necessary to study the long-range connections that exist between virtually all brain regions. This shortcoming led Bruno to develop a technique  for pair recording in vivo to examine the properties of synapses in the connections between thalamus and cortex (Fig. 6b,c) so as to test the relative importance of synaptic efficacy and population synchrony in tactile stimulus representation. Knowing the size of a unitary EPSP in a single VPM-to-L4 pair would allow one to set a minimum bound on how many thalamic APs mediate the compound PSP evoked in L4 spiny stellate cells by deflection of a single whisker. It constitutes a first step in delineating factors that contribute to the magnitude of stimulus representation and its trial-to-trial reliability in L4.
CNS synapses and cortical stimulus representation
One conclusion that can be drawn from examining two different types of synapses in vitro is that in the CNS, the functional properties of synapses, as described by n f, q and p r, vary widely. This differentiation, presumably, depends on the type of network to which the synapse belongs. It is therefore difficult, at present, to generalize the functional properties of a prototypical “CNS synapse” even if one restricts oneself to excitatory synapses with glutamate acting as a transmitter. Probably, functional properties of synapses are optimized by evolution and ontogeny within the context of their network function. Large functional changes could be the consequence of only minor changes in subcellular structure or in the composition of molecular components. A rather typical example for such subtle changes in structure that could have a large functional change would be an alteration of the kinetic properties of the Ca2+ sensor or its location in the AZ with respect to the sites of vesicular release. Unfortunately, CNS synapse phenomena, like plasticity and target cell specification, can be understood only by first measuring in detail their biophysical properties and in addition their likely contribution to network function.
Another conclusion related to this work is that the detailed biophysical characterization of synaptic efficacy in vitro must take into account the AP patterns of pre- and postsynaptic cells in vivo to disclose the values of n a, n f, p r, and q that are relevant for the understanding of CNS information processing, e.g., stimulus representation in different layers of cortex. These “effective” estimates could be very different in the intact animal, as suggested by the in vitro and in vivo experiments on the tc synapse. This seems to be particularly relevant for synapses at early stages of sensory pathways with a relatively high rate of spontaneously occurring APs and less for the intracortical pathways with their lower spontaneous AP rates.
Plasticity of synaptic efficacy in CNS synapses
Unbiased recording from cortical neurons in vivo  indicates sparse response in the upper layers of somatosensory cortex at least in anesthetized animals. Obviously, induction protocols for long-term changes in CNS synapses in vitro that use high-frequency presynaptic stimulation do not seem to be adequate for mimicking the in vivo situation at least in the cortex. Dual recording in vitro also has disclosed a possible new mechanism for the induction of long-term changes in synaptic efficacy of neocortical pathways . Here, the induction of mechanisms that change synaptic efficacy, which rely on coincidence of pre- and postsynaptic APs, may be more appropriate. One problem, however, is the low frequency of occurrence of coincident pre- and postsynaptic APs.
Column in silico project
The results of investigating pathway-specific synaptic properties mean that all connections (and their synapses) have to be studied as part of their natural network in vivo. This implies also that one has to know the AP pattern that in physiological conditions is “driving” synaptic transmission in a particular pathway. An extremely useful application of patch pipettes is the configuration of Pinault  of “juxtasomal recording” enabling the filling of cells, revealing their exact location in a pathway and their post hoc 3D reconstruction of dendrites and axon arbors. Eventually, a set of 3D reconstructions of neurons, e.g., in different layers of a column will have to be established in conjunction with their average response properties to sensory stimuli as well as an estimate of their average connectivity. Surprisingly, most studies on cortical representation still report extracellular recordings from morphologically unidentified neurons. Given the large variation of both the AP responses and the geometry of neurons in different cortical layers and even within a layer, it seems essential for the understanding of, e.g., sensory representation to classify cell types and their respective response properties more accurately. Only then can the wide spectrum of CNS synapses be studied biophysically in the more controlled in vivo conditions with naturally occurring AP patterns. On the other hand, various models of cortical representation or “coding” could profit from more realistic assumptions made about the particular cellular properties that determine the recorded AP patterns.
I would like to thank R. Bruno and E. Heil for their help in preparing this manuscript. I also thank G. Borst (Rotterdam), T. Kuner, C. de Kock, and H. Spors (Heidelberg) for the discussions, and D. Bannerman (Oxford) for the corrections in the text.
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