Unitary Excitatory Postsynaptic Potentials Research Articles

Shared and divergent principles of synaptic transmission between cortical excitatory neurons in rodent and human brain.

Information transfer between principal neurons in neocortex occurs through (glutamatergic) synaptic transmission. In this focussed review, we provide a detailed overview on the strength of synaptic neurotransmission between pairs of excitatory neurons in human and laboratory animals with a specific focus on data obtained using patch clamp electrophysiology. We reach two major conclusions: (1) the synaptic strength, measured as unitary excitatory postsynaptic potential (or uEPSP), is remarkably consistent across species, cortical regions, layers and/or cell-types (median 0.5 mV, interquartile range 0.4-1.0 mV) with most variability associated with the cell-type specific connection studied (min 0.1-max 1.4 mV), (2) synaptic function cannot be generalized across human and rodent, which we exemplify by discussing the differences in anatomical and functional properties of pyramidal-to-pyramidal connections within human and rodent cortical layers 2 and 3. With only a handful of studies available on synaptic transmission in human, it is obvious that much remains unknown to date. Uncovering the shared and divergent principles of synaptic transmission across species however, will almost certainly be a pivotal step toward understanding human cognitive ability and brain function in health and disease.

Strong and reliable synaptic communication between pyramidal neurons in adult human cerebral cortex.

Synaptic transmission constitutes the primary mode of communication between neurons. It is extensively studied in rodent but not human neocortex. We characterized synaptic transmission between pyramidal neurons in layers 2 and 3 using neurosurgically resected human middle temporal gyrus (MTG, Brodmann area 21), which is part of the distributed language circuitry. We find that local connectivity is comparable with mouse layer 2/3 connections in the anatomical homologue (temporal association area), but synaptic connections in human are 3-fold stronger and more reliable (0% vs 25% failure rates, respectively). We developed a theoretical approach to quantify properties of spinous synapses showing that synaptic conductance and voltage change in human dendritic spines are 3-4-folds larger compared with mouse, leading to significant NMDA receptor activation in human unitary connections. This model prediction was validated experimentally by showing that NMDA receptor activation increases the amplitude and prolongs decay of unitary excitatory postsynaptic potentials in human but not in mouse connections. Since NMDA-dependent recurrent excitation facilitates persistent activity (supporting working memory), our data uncovers cortical microcircuit properties in human that may contribute to language processing in MTG.

Olfaction in Lamprey Pallium Revisited—Dual Projections of Mitral and Tufted Cells

The presence of two separate afferent channels from the olfactory glomeruli to different targets in the brain is unravelled in the lamprey. The mitral-like cells send axonal projections directly to the piriform cortex in the ventral part of pallium, whereas the smaller tufted-like cells project separately and exclusively to a relay nucleus called the dorsomedial telencephalic nucleus (dmtn). This nucleus, located at the interface between the olfactory bulb and pallium, in turn projects to a circumscribed area in the anteromedial, ventral part of pallium. The tufted-like cells are activated with short latency from the olfactory nerve and terminate with mossy fibers on the dmtn cells, wherein they elicit large unitary excitatory postsynaptic potentials (EPSPs). In all synapses along this tufted-like cell pathway, there is no concurrent inhibition, in contrast to the mitral-like cell pathway. This is similar to recent findings in rodents establishing two separate exclusive projection patterns, suggesting an evolutionarily conserved organization.

The intrinsic cell type-specific excitatory connectivity of the developing mouse subiculum is sufficient to generate synchronous epileptiform activity.

The activity of local excitatory circuits of the subiculum has been suggested to be involved in the initiation of pathological activity in epileptic patients and experimental animal models of temporal lobe epilepsy. We have taken advantage of multimodal techniques to classify subicular cells in distinct subclasses and have investigated their morphofunctional properties and connectivity in vitro. Our results indicate that local subicular excitatory circuits are connected in a cell type-specific fashion and that synapses are preferentially established on basal vs. apical dendrites. We show that local excitatory circuits, isolated from extrasubicular inputs and pharmacologically disinhibited, are sufficient to initiate synchronous epileptiform activity in vitro. In conclusion, this work provides a high-resolution description of local excitatory circuits of the subiculum and highlights their mechanistic involvement in the generation of pathological activity. The subiculum has been suggested to be involved in the initiation of pathological discharges in human patients and animal models of temporal lobe epilepsy. Although converging evidence has revealed the existence of functional diversity within its principal neurons, much less attention has been devoted to its intrinsic connectivity and whether its local excitatory circuits are sufficient to generate epileptiform activity. Here, we have directly addressed these two key points in mouse subicular slices. First, using multivariate techniques, we have distinguished two groups of principal cells, which we have termed type1 and type2. These subgroups roughly overlap with what were classically indicated as regular and bursting cells, and showed differences in the extension and complexity of their apical dendrites. Functional connectivity was found both between similar (homotypic) and different (heterotypic) types of cells, with a marked asymmetry within heterotypic pairs. Unitary excitatory postsynaptic potentials (uEPSPs) revealed a high degree of variability both in amplitude, failure rate, rise time and half-width. Post hoc analysis of functionally connected pairs suggested that the observed uEPSPs were mediated by few contact sites, predominantly located on the basal dendrites. When surgically isolated from extrasubicular excitatory afferents, pharmacologically disinhibited subicular slices generated hyper-synchronous discharges. Thus, we conclude that local subicular excitatory circuits, connected according to cell type-specific rules, are sufficient to promote epileptiform activity. This conclusion fits well with a previous suggestion that local subicular events, purely mediated by excitatory connections, may underlie the pre-ictal discharges that govern interictal-ictal transitions.

Recurrent connections between CA2 pyramidal cells.

Recurrent excitatory synapses have theoretically been shown to play roles in memory storage and associative learning and are well described to occur in the CA3 region of the hippocampus. Here, we report that the CA2 region also contains recurrent excitatory monosynaptic couplings. Using dual whole-cell patch-clamp recordings from CA2 pyramidal cells in mouse hippocampal slices under differential interference contrast microscopic controls, we evaluated monosynaptic excitatory connections. Unitary excitatory postsynaptic potentials occurred in 1.4% of 502 cell pairs. These connected pairs were preferentially located in the superficial layer and proximal part (CA2b) of the CA2 region. These results indicate that recurrent excitatory circuits are denser in the CA2 region than in the CA1 region, as well as in the CA3 region.

State-dependent cell-type-specific membrane potential dynamics and unitary synaptic inputs in awake mice.

The cellular and synaptic mechanisms driving cell-type-specific function during various cortical network activities and behaviors are poorly understood. Here, we targeted whole-cell recordings to two classes of inhibitory GABAergic neurons in layer 2/3 of the barrel cortex of awake head-restrained mice and correlated spontaneous membrane potential dynamics with cortical state and whisking behavior. Using optogenetic stimulation of single layer 2/3 excitatory neurons we measured unitary excitatory postsynaptic potentials (uEPSPs) across states. During active states, characterized by whisking and reduced low-frequency activity in the local field potential, parvalbumin-expressing neurons depolarized and, albeit in a small number of recordings, received uEPSPs with increased amplitude. In contrast, somatostatin-expressing neurons hyperpolarized and reduced firing rates during active states without consistent change in uEPSP amplitude. These results further our understanding of neocortical inhibitory neuron function in awake mice and are consistent with the hypothesis that distinct genetically-defined cell classes have different state-dependent patterns of activity.

Plasticity-dependent, full detonation at hippocampal mossy fiber-CA3 pyramidal neuron synapses.

Mossy fiber synapses on CA3 pyramidal cells are 'conditional detonators' that reliably discharge postsynaptic targets. The 'conditional' nature implies that burst activity in dentate gyrus granule cells is required for detonation. Whether single unitary excitatory postsynaptic potentials (EPSPs) trigger spikes in CA3 neurons remains unknown. Mossy fiber synapses exhibit both pronounced short-term facilitation and uniquely large post-tetanic potentiation (PTP). We tested whether PTP could convert mossy fiber synapses from subdetonator into detonator mode, using a recently developed method to selectively and noninvasively stimulate individual presynaptic terminals in rat brain slices. Unitary EPSPs failed to initiate a spike in CA3 neurons under control conditions, but reliably discharged them after induction of presynaptic short-term plasticity. Remarkably, PTP switched mossy fiber synapses into full detonators for tens of seconds. Plasticity-dependent detonation may be critical for efficient coding, storage, and recall of information in the granule cell-CA3 cell network.

Serotonin modulates the excitatory synaptic transmission in the dentate granule cells.

Serotonergic fibers from the raphe nuclei project to the hippocampal formation, the activity of which is known to modulate the inhibitory interneurons in the dentate gyrus. On the other hand, serotonergic modulation of the excitatory synapses in the dentate gyrus is not well examined. In the present study, we examined the effects of 5-HT on the excitatory postsynaptic potentials (EPSPs) in the dentate granule cells evoked by the selective stimulation of the lateral perforant path (LPP), the medial perforant path (MPP), or the mossy cell fibers (MCF). 5-HT depressed the amplitude of unitary EPSPs (uEPSPs) evoked by the stimulation of LPP or MPP, whereas uEPSPs evoked by MCF stimulation were little affected. The effect was partly explained by the decrease of the resting membrane resistance following the activation of 5-HT1A receptors, which was confirmed by computer simulations. We also found that the probability of evoking uEPSP by LPP stimulation but not MPP or MCF stimulation was reduced by 5-HT and that the paired-pulse ratio of LPP-evoked EPSP but not that of MPP- or MCF-evoked ones was increased by 5-HT. These effects were blocked by 5-HT2 antagonist, suggesting that the transmitter release in the LPP-granule cell synapse is inhibited by the activation of 5-HT2 receptors. The present results suggest that 5-HT can modulate the EPSPs in the dentate granule cells by at least two distinct mechanisms.

In Vivo Monosynaptic Excitatory Transmission between Layer 2 Cortical Pyramidal Neurons.

SummaryLittle is known about the properties of monosynaptic connections between identified neurons in vivo. We made multiple (two to four) two-photon targeted whole-cell recordings from neighboring layer 2 mouse somatosensory barrel cortex pyramidal neurons in vivo to investigate excitatory monosynaptic transmission in the hyperpolarized downstate. We report that pyramidal neurons form a sparsely connected (6.7% connectivity) network with an overrepresentation of bidirectional connections. The majority of unitary excitatory postsynaptic potentials were small in amplitude (<0.5 mV), with a small minority >1 mV. The coefficient of variation (CV = 0.74) could largely be explained by the presence of synaptic failures (22%). Both the CV and failure rates were reduced with increasing amplitude. The mean paired-pulse ratio was 1.15 and positively correlated with the CV. Our approach will help bridge the gap between connectivity and function and allow investigations into the impact of brain state on monosynaptic transmission and integration.

Dendritic Target Region-Specific Formation of Synapses Between Excitatory Layer 4 Neurons and Layer 6 Pyramidal Cells.

Excitatory connections between neocortical layer 4 (L4) and L6 are part of the corticothalamic feedback microcircuitry. Here we studied the intracortical element of this feedback loop, the L4 spiny neuron-to-L6 pyramidal cell connection. We found that the distribution of synapses onto both putative corticothalamic (CT) and corticocortical (CC) L6 pyramidal cells (PCs) depends on the presynaptic L4 neuron type but is independent of the postsynaptic L6 PC type. L4 spiny stellate cells establish synapses on distal apical tuft dendrites of L6 PCs and elicit slow unitary excitatory postsynaptic potentials (uEPSPs) in L6 somata. In contrast, the majority of L4 star pyramidal neurons target basal and proximal apical oblique dendrites of L6 PCs and show fast uEPSPs. Compartmental modeling suggests that the slow uEPSP time course is primarily the result of dendritic filtering. This suggests that the dendritic target specificity of the 2 L4 spiny neuron types is due to their different axonal projection patterns across cortical layers. The preferential dendritic targeting by different L4 neuron types may facilitate the generation of dendritic Ca(2+) or Na(+) action potentials in L6 PCs; this could play a role in synaptic gain modulation in the corticothalamic pathway.

Nonlinear AMPA Sensitivity to Glutamate and Recruitment of NMDA Activity in Dendritic Spines

Our lab has been developing and improving the optical and molecular tools and techniques to record EPSPs (excitatory postsynaptic potentials) in dendritic spines. With a dual laser 2-photon microscope we are able to record from voltage-sensitive dye (VSD) labeled spines from cortical pyramidal neurons while evoking unitary EPSPs using glutamate uncaging. By varying the uncaging laser intensity we observe a nonlinear response to glutamate when the resulting EPSPs amplitudes are observed at the soma. By optically recording EPSPs in spines we can determine that the observed nonlinearity is often present even when spine EPSPs are relatively small, i.e. < 10 mV. Pharmacology is used to block NMDA receptors and determine the recruitment of these receptors and their contribution to both voltage and calcium influx. Calcium imaging reveals that NMDA receptors are recruited in a very linear fashion according to the spine depolarization. This calcium influx however, does not appear to play a significant role in the spine depolarization. Finally, modeling is used to reconcile the observed phenomena with detailed models of AMPA and NMDA receptor gating, and glutamate binding.

In Vivo Measurement of Cell-Type-Specific Synaptic Connectivity and Synaptic Transmission in Layer 2/3 Mouse Barrel Cortex

SummaryIntracellular recordings of membrane potential in vitro have defined fundamental properties of synaptic communication. Much less is known about the properties of synaptic connectivity and synaptic transmission in vivo. Here, we combined single-cell optogenetics with whole-cell recordings to investigate glutamatergic synaptic transmission in vivo from single identified excitatory neurons onto two genetically defined subtypes of inhibitory GABAergic neurons in layer 2/3 mouse barrel cortex. We found that parvalbumin-expressing (PV) GABAergic neurons received unitary glutamatergic synaptic input with higher probability than somatostatin-expressing (Sst) GABAergic neurons. Unitary excitatory postsynaptic potentials onto PV neurons were also faster and more reliable than inputs onto Sst neurons. Excitatory synapses targeting Sst neurons displayed strong short-term facilitation, while those targeting PV neurons showed little short-term dynamics. Our results largely agree with in vitro measurements. We therefore demonstrate the technical feasibility of assessing functional cell-type-specific synaptic connectivity in vivo, allowing future investigations into context-dependent modulation of synaptic transmission.

Microcircuits of excitatory and inhibitory neurons in layer 2/3 of mouse barrel cortex

Synaptic interactions between nearby excitatory and inhibitory neurons in the neocortex are thought to play fundamental roles in sensory processing. Here, we have combined optogenetic stimulation, whole cell recordings, and computational modeling to define key functional microcircuits within layer 2/3 of mouse primary somatosensory barrel cortex. In vitro optogenetic stimulation of excitatory layer 2/3 neurons expressing channelrhodopsin-2 evoked a rapid sequence of excitation followed by inhibition. Fast-spiking (FS) GABAergic neurons received large-amplitude, fast-rising depolarizing postsynaptic potentials, often driving action potentials. In contrast, the same optogenetic stimulus evoked small-amplitude, subthreshold postsynaptic potentials in excitatory and non-fast-spiking (NFS) GABAergic neurons. To understand the synaptic mechanisms underlying this network activity, we investigated unitary synaptic connectivity through multiple simultaneous whole cell recordings. FS GABAergic neurons received unitary excitatory postsynaptic potentials with higher probability, larger amplitudes, and faster kinetics compared with NFS GABAergic neurons and other excitatory neurons. Both FS and NFS GABAergic neurons evoked robust inhibition on postsynaptic layer 2/3 neurons. A simple computational model based on the experimentally determined electrophysiological properties of the different classes of layer 2/3 neurons and their unitary synaptic connectivity accounted for key aspects of the network activity evoked by optogenetic stimulation, including the strong recruitment of FS GABAergic neurons acting to suppress firing of excitatory neurons. We conclude that FS GABAergic neurons play an important role in neocortical microcircuit function through their strong local synaptic connectivity, which might contribute to driving sparse coding in excitatory layer 2/3 neurons of mouse barrel cortex in vivo.

Parvalbumin‐producing cortical interneurons receive inhibitory inputs on proximal portions and cortical excitatory inputs on distal dendrites

To examine inputs to parvalbumin (PV)-producing interneurons, we generated transgenic mice expressing somatodendritic membrane-targeted green fluorescent protein specifically in the interneurons, and completely visualized their dendrites and somata. Using immunolabeling for vesicular glutamate transporter (VGluT)1, VGluT2, and vesicular GABA transporter, we found that VGluT1-positive terminals made contacts 4- and 3.1-fold more frequently with PV-producing interneurons than VGluT2-positive and GABAergic terminals, respectively, in the primary somatosensory cortex. Even in layer 4, where VGluT2-positive terminals were most densely distributed, VGluT1-positive inputs to PV-producing interneurons were 2.4-fold more frequent than VGluT2-positive inputs. Furthermore, although GABAergic inputs to PV-producing interneurons were as numerous as VGluT2-positive inputs in most cortical layers, GABAergic inputs clearly preferred the proximal dendrites and somata of the interneurons, indicating that the sites of GABAergic inputs were more optimized than those of VGluT2-positive inputs. Simulation analysis with a PV-producing interneuron model compatible with the present morphological data revealed a plausible reason for this observation, by showing that GABAergic and glutamatergic postsynaptic potentials evoked by inputs to distal dendrites were attenuated to 60 and 87%, respectively, of those evoked by somatic inputs. As VGluT1-positive and VGluT2-positive axon terminals were presumed to be cortical and thalamic glutamatergic inputs, respectively, cortical excitatory inputs to PV-producing interneurons outnumbered the thalamic excitatory and intrinsic inhibitory inputs more than two-fold in any cortical layer. Although thalamic inputs are known to evoke about two-fold larger unitary excitatory postsynaptic potentials than cortical ones, the present results suggest that cortical inputs control PV-producing interneurons at least as strongly as thalamic inputs.

Short-term modulation at synapses between neurons in laminae II–V of the rodent spinal dorsal horn

Unitary excitatory (EPSP) and inhibitory (IPSP) postsynaptic potentials (PSPs) were evoked between neurons in Rexed's laminae (L)II-V of spinal slices from young hamsters (7-24 days old) at 27°C using paired whole cell recordings. Laminar differences in synaptic efficacy were observed: excitatory connections were more secure than inhibitory connections in LII and inhibitory linkages in LII were less reliable than those in LIII-V. A majority of connections displayed paired-pulse facilitation or depression. Depression was observed for both EPSPs and IPSPs, but facilitation was seen almost exclusively for IPSPs. There were no frequency-dependent shifts between facilitation and depression. Synaptic depression was associated with an increased failure rate and decreased PSP half-width for a majority of connections. However, there were no consistent changes in failure rate or PSP time course at facilitating connections. IPSPs evoked at high-failure synapses had consistently smaller amplitude and showed greater facilitation than low-failure connections. Facilitation at inhibitory connections was positively correlated with synaptic jitter and associated with a decrease in latency. At many connections, the paired-pulse ratio varied from trial to trial and depended on the amplitude of the first PSP; dependence was greater for inhibitory synapses than excitatory synapses. Paired-pulse ratios for connections onto neurons with rapidly adapting, "phasic" discharge to depolarizing current injection were significantly greater than for connections onto neurons with tonic discharge properties. These results are evidence of diversity in synaptic transmission between dorsal horn neurons, the nature of which may depend on the types of linkage, laminar location, and intrinsic firing properties of postsynaptic cells.

Cellular and Synaptic Actions of Acetylcholine in the Lamprey Spinal Cord

This study investigated cellular and synaptic mechanisms of cholinergic neuromodulation in the in vitro lamprey spinal cord. Most spinal neurons tested responded to local application of acetylcholine (ACh) with depolarization and decreased input resistance. The depolarization persisted in the presence of either tetrodotoxin or muscarinic antagonist scopolamine and was abolished with nicotinic antagonist mecamylamine, indicating a direct depolarization through nicotinic ACh receptors. Local application of muscarinic ACh agonists modulated synaptic strength in the spinal cord by decreasing the amplitude of unitary excitatory and inhibitory postsynaptic potentials. The postsynaptic response to direct application of glutamate was unchanged by muscarinic agonists, suggesting a presynaptic mechanism. Cholinergic feedback from motoneurons was assessed using stimulation of a ventral root in the quiescent spinal cord while recording intracellularly from spinal motoneurons or interneurons. Mainly depolarizing potentials were observed, a portion of which was insensitive to removal of extracellular Ca2+, indicating electrotonic coupling. Hyperpolarizing potentials were also observed and were attenuated by the glycinergic antagonist strychnine, whereas depolarizing responses were potentiated by strychnine. Mecamylamine also reduced hyperpolarizing responses. The pharmacology of these responses suggests a Renshaw-like feedback pathway in lamprey. Immunohistochemistry for choline acetyltransferase, performed in combination with retrograde filling of motoneurons, demonstrated a population of nonmotoneuron cholinergic cells in the lamprey spinal cord. Thus endogenous cholinergic modulation of the lamprey spinal locomotor network is likely produced by both motoneurons and cholinergic interneurons acting via combined postsynaptic and presynaptic actions.

Functional studies of motor cortex.

Defining functions for neural elements becomes more difficult the more remote they are, in synaptic linkages, from motor neurons. The precentral motor cortex contains a corticomotoneuronal projection system, only one synapse removed from motor neurons. Corticomotoneuronal fibres produce monosynaptic excitation of spinal motor neurons, which is more powerful for those acting distally, innervating extensor muscles of the fingers and intrinsic hand muscles. Latencies and time-course of corticomotoneuronal excitation are defined. Amplitudes of unitary corticomotoneuronal excitatory postsynaptic potentials are very small. Many corticomotoneuronal cells converge on a given motor neuron. Some motor neurons innervating proximally acting muscles appear to receive no corticomotoneuronal excitation. Disynaptic inhibitory actions are produced by corticospinal volleys via the common Ia inhibitory interneuron--possibly reciprocal actions produced over collaterals of corticomotoneuronal fibres. Anatomical divergence of projections of collaterals of an identified corticomotoneuronal fibre is extensive enough to provide both for delivery of synapses to a large number of motor neurons and for the dispersion of specific projections to inhibitory interneurons, to fusimotor neurons and to other interneurons. Functions of corticomotoneuronal elements in motor cortex whose targets have been identified and whose excitatory or inhibitory actions have been specified by cross-correlation have been studied by Fetz & Cheney (J Neurophysiol 1980; 44:751-772).

Monosynaptic Connections between Pairs of L5A Pyramidal Neurons in Columns of Juvenile Rat Somatosensory Cortex

Layer 5 (L5) of somatosensory cortex is a major gateway for projections to intra- and subcortical brain regions. This layer is further divided into 5A and 5B characterized by relatively separate afferent and efferent connections. Little is known about the organization of connections within L5A of neocortical columns. We therefore used paired recordings to probe the anatomy and physiology of monosynaptic connections between L5A pyramidal neurons within the barrel columns of somatosensory cortex in acute slices of approximately 3-week-old rats. Post hoc reconstruction and calculation of the axodendritic overlap of pre- and postsynaptic neurons, together with identification of putative synaptic contacts (3.5 per connection), indicated a preferred innervation domain in the proximal dendritic region. Synaptic transmission was reliable (failure rate <2%) and had a low variability (coefficient of variation of 0.3). Unitary excitatory postsynaptic potential (EPSP) amplitudes varied 30-fold with a mean of 1.2 mV and displayed depression over a wide range of frequencies (2-100 Hz) during bursts of presynaptic firing. A single L5A pyramidal neuron was estimated to target approximately 270 other pyramidal neurons within the same layer of its home barrel column, suggesting a mechanism of feed-forward excitation by which synchronized single action potentials are efficiently transmitted within L5A of juvenile cortex.

Take Your PIC: Motoneuronal Persistent Inward Currents May Be Somatic as Well as Dendritic. Focus on “Facilitation of Somatic Calcium Channels Can Evoke Prolonged Tail Currents in Rat Hypoglossal Motoneurons”

Inputs to motoneurons can be amplified and prolonged by noninactivating conductances mediating “persistent inward currents” or PICs. These currents, first identified by [Schwindt and Crill (1977)][1], are generated through voltage-dependent channels that have been thought to be confined to the

Respiratory drive to thoracic motoneurones

The paper by Saywell et al. (2007) in this issue of The Journal of Physiology provides a detailed study of the projection of the expiratory bulbospinal neurones to identified thoracic motoneurones in the anaesthetized cat. The importance of the paper lies first in its clarification of the debate regarding the strength of the monosynaptic excitation from this pathway and secondly, the deduction of the significance of the pathway in driving natural rhythmic motoneurone firing. The methods developed and the conclusions derived are relevant not only to the special case of respiration, but also to the more general consideration of the part played by particular sources of excitation in generating motoneurone firing patterns in other systems. It has long been known that both the inspiratory and the expiratory phases of breathing are dependent on activity in groups of cells in the hindbrain (Pitts et al. 1939). The most extensively studied group are in the nucleus retroambigualis and their output is carried to various motoneurones involved in respiration via crossed bulbospinal fibres. Previous studies have indicated that both phrenic and inspiratory intercostal motoneurones receive monosynaptic excitation from this source, but probably depend to a large degree on multisynaptic connections for their natural firing pattern (Davies et al. 1985). The relative contributions of mono- and multisynaptic connections to expiratory motoneurones, on the other hand, have not been clear. Using intracellular spike-triggered averaging (STA) from expiratory bulbospinal neurones (EBSNs) to internal intercostal motoneurones Merrill & Lipski (1987) found only very few and weak monosynaptic excitatory postsynaptic potentials (EPSPs), which could account for neither the rhythmic motoneurone firing nor the observed central respiratory drive potentials (CRDPs). Cross-correlation of EBSN spikes with expiratory nerve filaments also rarely showed the short duration peak expected for a monosynaptic connection. It was surprising therefore that when a spike correlation study was repeated by Kirkwood (1995) most EBSN neurones showed clear evidence of monosynaptic projection, which was estimated to be producing most of the depolarization required to drive the rhythmic firing of the expiratory motoneurones. A valuable review of this background was provided by Kirkwood et al. (1999) together with preliminary data now reported in detail. This new study has confirmed, by intracellular STA, the presence of a potent monosynaptic connection, especially to expiratory neurones with their axons in the internal intercostal nerves. Computations, taking account of directly measured EPSP duration and amplitude and reasonable estimates of numbers of EBSNs and of their firing frequencies, indicate that the sum of the unitary EPSPs could account for the observed CRDPs. Indeed the data imply a projection at least 10 times as strong as that found by Merrill & Lipski (1987). Of course the question arises as to how there can be such a disparity in data from basically similar experiments. Factors such as bias in selection of motoneurone types, rostral or caudal recording sites in a segment or variation according to the part of the thorax supplied were all considered but rejected. A final possibility arises from the observation of a correlation between EPSP size and CRDP amplitude, which suggests that some state-dependent modulation in EPSP amplitude may vary between different preparations, for example as a result of differing anaesthesia. Although the same agent (pentobarbitone) was employed in both cases, Saywell et al. (2007) appear to have controlled the dosage to give a lighter level. Nevertheless, this seems unlikely to be a very important factor and other causes should be considered. It is worth recalling the observations of Nelson et al. (1979) showing acute increases in Ia EPSP amplitudes following spinal transection. The mechanism of such state-dependent modulation is not considered, but two factors might be examined in future work. There could well be a presynaptic inhibitory mechanism involved (see Rudomin & Schmidt, 1999) which could vary due to small differences in the preparation. Also, the incidence of failures of transmission (Grimwood et al. 1992) could differ according to the condition of the animal. It is interesting to speculate as to the functional significance of a potentially strong monosynaptic EBSN projection to expiratory motoneurones. We have come to associate monosynaptic descending control with the need for precision, as in hand movements in primates. Perhaps similar precision may be required in some expiratory movements such as vocalization (Sears, 1977), though cats are not noted for their musical skills. The more serious message is the importance of detailed quantitative studies, such as reported by Saywell et al. (2007) in establishing the relative significance for normal function of various neural projections, which are generally only established qualitatively.