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In many neurological conditions, early-stage neural circuit adaption can preserve relatively normal behaviour. In some diseases, spinal motoneurons progressively degenerate yet movement is initially preserved. We therefore investigated whether these neurons and associated microcircuits adapt in a mouse model of progressive motoneuron degeneration. Using a combination of in vitro and in vivo electrophysiology and super-resolution microscopy, we found that, early in the disease, neurotransmission in a key pre-motor circuit, the recurrent inhibition mediated by Renshaw cells, is reduced by half due to impaired quantal size associated with decreased glycine receptor density. This impairment is specific, and not a widespread feature of spinal inhibitory circuits. Furthermore, it recovers at later stages of disease. Additionally, an increased probability of release from proprioceptive afferents leads to increased monosynaptic excitation of motoneurons. We reveal that in motoneuron degenerative conditions, spinal microcircuits undergo specific multiphasic homeostatic compensations that may contribute to preservation of force output., Competing Interests: Declaration of interests R.M.B. is a co-founder and is on the board of Sania Therapeutics Inc. and consults for Sania Rx Ltd.

Elaborate behaviours are produced by tightly controlled flexor-extensor motor neuron activation patterns. Motor neurons are regulated by a network of interneurons within the spinal cord, but the computational processes involved in motor control are not fully understood. The neuroanatomical arrangement of motor and premotor neurons into topographic patterns related to their controlled muscles is thought to facilitate how information is processed by spinal circuits. Rabies retrograde monosynaptic tracing has been used to label premotor interneurons innervating specific motor neuron pools, with previous studies reporting topographic mediolateral positional biases in flexor and extensor premotor interneurons. To more precisely define how premotor interneurons contacting specific motor pools are organized, we used multiple complementary viral-tracing approaches in mice to minimize systematic biases associated with each method. Contrary to expectations, we found that premotor interneurons contacting motor pools controlling flexion and extension of the ankle are highly intermingled rather than segregated into specific domains like motor neurons. Thus, premotor spinal neurons controlling different muscles process motor instructions in the absence of clear spatial patterns among the flexor-extensor circuit components., Competing Interests: RR, SS, BB, BB, GB, JF, JM, CL, AP, MÖ, AT, DM, SP, NZ, MB No competing interests declared, CS is currently an employee of Sania therapeutics. His work is not related to that reported in this manuscript, AM is a co-founder of Sania Therapeutics. His work is not related to that reported in this manuscript. AM has founders stock in Sania Therapeutics, Ltd, RB has a grant from Medical Research Council, is a a co-founder of the parent company, Sania Therapeutics, Ltd. Work not related to that reported in this manuscript. RB is as an Associate Editor for Journal of Neurophysiology, has a patent (18 July, 2020, Priority patent GB 2010981.5; published 20 Jan, 2022, WO/2022/013396 "Gene therapy for neuromuscular and neuromotor disorders") unrelated to this manuscript and is an unpaid Trustee for Stoke Mandeville Spinal Research, International Spinal Research Trust and Scientific Advisory Board, Brain Research UK. RB has founders stock in Sania Therapeutics, Ltd, (© 2022, Ronzano, Skarlatou, Barriga et al.)

Motoneurons (MNs) control muscle contractions, and their recruitment by premotor circuits is tuned to produce accurate motor behaviours. To understand how these circuits coordinate movement across and between joints, it is necessary to understand whether spinal neurons pre-synaptic to motor pools have divergent projections to more than one MN population. Here, we used modified rabies virus tracing in mice to investigate premotor interneurons projecting to synergist flexor or extensor MNs, as well as those projecting to antagonist pairs of muscles controlling the ankle joint. We show that similar proportions of premotor neurons diverge to synergist and antagonist motor pools. Divergent premotor neurons were seen throughout the spinal cord, with decreasing numbers but increasing proportion with distance from the hindlimb enlargement. In the cervical cord, divergent long descending propriospinal neurons were found in contralateral lamina VIII, had large somata, were neither glycinergic, nor cholinergic, and projected to both lumbar and cervical MNs. We conclude that distributed spinal premotor neurons coordinate activity across multiple motor pools and that there are spinal neurons mediating co-contraction of antagonist muscles., Competing Interests: RR, CL, GB, RB, MB No competing interests declared, (© 2021, Ronzano et al.)

Parvalbumin (PV)-positive interneurons modulate cortical activity through highly specialized connectivity patterns onto excitatory pyramidal neurons (PNs) and other inhibitory cells. PV cells are autoconnected through powerful autapses, but the contribution of this form of fast disinhibition to cortical function is unknown. We found that autaptic transmission represents the most powerful inhibitory input of PV cells in neocortical layer V. Autaptic strength was greater than synaptic strength onto PNs as a result of a larger quantal size, whereas autaptic and heterosynaptic PV-PV synapses differed in the number of release sites. Overall, single-axon autaptic transmission contributed to approximately 40% of the global inhibition (mostly perisomatic) that PV interneurons received. The strength of autaptic transmission modulated the coupling of PV-cell firing with optogenetically induced γ-oscillations, preventing high-frequency bursts of spikes. Autaptic self-inhibition represents an exceptionally large and fast disinhibitory mechanism, favoring synchronization of PV-cell firing during cognitive-relevant cortical network activity., Competing Interests: The authors have declared that no competing interests exist.

Spinal motoneurones (Mns) constitute the final output for the execution of motor tasks. In addition to innervating muscles, Mns project excitatory collateral connections to Renshaw cells (RCs) and other Mns, but the latter have received little attention. We show that Mns receive strong synaptic input from other Mns throughout development and into maturity, with fast-type Mns systematically receiving greater recurrent excitation than slow-type Mns. Optical recordings show that activation of Mns in one spinal segment can propagate to adjacent segments even in the presence of intact recurrent inhibition. While it is known that transmission at the neuromuscular junction is purely cholinergic and RCs are excited through both acetylcholine and glutamate receptors, here we show that neurotransmission between Mns is purely glutamatergic, indicating that synaptic transmission systems are differentiated at different postsynaptic targets of Mns.

In neonatal mice motoneurons excite Renshaw cells by releasing both acetylcholine (ACh) and glutamate. These two neurotransmitters activate two types of nicotinic receptors (nAChRs) (the homomeric α 7 receptors and the heteromeric α*ß* receptors) as well as the two types of glutamate receptors (GluRs) (AMPARs and NMDARs). Using paired recordings, we confirm that a single motoneuron can release both transmitters on a single post-synaptic Renshaw cell. We then show that co-transmission is preserved in adult animals. Kinetic analysis of miniature EPSCs revealed quantal release of mixed events associating AMPARs and NMDARs, as well as α 7 and α*ß* nAChRs, but no evidence was found for mEPSCs associating nAChRs with GluRs. Bayesian Quantal Analysis (BQA) of evoked EPSCs showed that the number of functional contacts on a single Renshaw cell is more than halved when the nicotinic receptors are blocked, confirming that the two neurotransmitters systems are segregated. Our observations can be explained if ACh and glutamate are released from common vesicles onto spatially segregated post-synaptic receptors clusters, but a pre-synaptic segregation of cholinergic and glutamatergic release sites is also possible.

Renshaw cells represent a fundamental component of one of the first discovered neuronal circuits, but their function in motor control has not been established. They are the only central neurons that receive collateral projections from motor outputs, yet the efficacy of the excitatory synapses from single and converging motoneurons remains unknown. Here we present the results of dual whole-cell recordings from identified, synaptically connected Renshaw cell-motoneuron pairs in the mouse lumbar spinal cord. The responses from single Renshaw cells demonstrate that motoneuron synapses elicit large excitatory conductances with few or no failures. We show that the strong excitatory input from motoneurons results from a high probability of neurotransmitter release onto multiple postsynaptic contacts. Dual current-clamp recordings confirm that single motoneuron inputs were sufficient to depolarize the Renshaw cell beyond threshold for firing. Reciprocal connectivity was observed in approximately one-third of the paired recordings tested. Ventral root stimulation was used to evoke currents from Renshaw cells or motoneurons to characterize responses of single neurons to the activation of their corresponding presynaptic cell populations. Excitatory or inhibitory synaptic inputs in the recurrent inhibitory loop induced substantial effects on the excitability of respective postsynaptic cells. Quantal analysis estimates showed a large number of converging inputs from presynaptic motoneuron and Renshaw cell populations. The combination of considerable synaptic efficacy and extensive connectivity within the recurrent circuitry indicates a role of Renshaw cells in modulating motor outputs that may be considerably more important than has been previously supposed., Significance Statement: We have recently shown that Renshaw cells mediate powerful shunt inhibition on motoneuron excitability. Here we complete a quantitative description of the recurrent circuit using recordings of excitatory synapses between identified motoneuron and Renshaw cell pairs. We show that the excitation is highly effective as a result of a high probability of neurotransmitter release onto multiple release sites and that efficient neurotransmission is maintained at physiologically relevant firing rates in motoneurons. Our results also show that both excitatory and inhibitory connections exhibit considerable convergence of inputs. Because evaluation of the determinants of synaptic strength and the extent of connectivity constitute fundamental parameters affecting the operation of the recurrent circuit, our findings are critical for informing any future models of motor control., (Copyright © 2015 Moore, Bhumbra et al.)

Although Renshaw cells (RCs) were discovered over half a century ago, their precise role in recurrent inhibition and ability to modulate motoneuron excitability have yet to be established. Indirect measurements of recurrent inhibition have suggested only a weak modulatory effect but are limited by the lack of observed motoneuron responses to inputs from single RCs. Here we present dual recordings between connected RC-motoneuron pairs, performed on mouse spinal cord. Motoneuron responses demonstrated that Renshaw synapses elicit large inhibitory conductances and show short-term potentiation. Anatomical reconstruction, combined with a novel method of quantal analysis, showed that the strong inhibitory input from RCs results from the large number of synaptic contacts that they make onto individual motoneurons. We used the NEURON simulation environment to construct realistic electrotonic models, which showed that inhibitory conductances from Renshaw inputs exert considerable shunting effects in motoneurons and reduce the frequency of spikes generated by excitatory inputs. This was confirmed experimentally by showing that excitation of a single RC or selective activation of the recurrent inhibitory pathway to generate equivalent inhibitory conductances both suppress motoneuron firing. We conclude that recurrent inhibition is remarkably effective, in that a single action potential from one RC is sufficient to silence a motoneuron. Although our results may differ from previous indirect observations, they underline a need for a reevaluation of the role that RCs perform in one of the first neuronal circuits to be discovered., (Copyright © 2014 Bhumbra et al.)

Communication between neurones in the central nervous system depends on synaptic transmission. The efficacy of synapses is determined by pre- and postsynaptic factors that can be characterized using quantal parameters such as the probability of neurotransmitter release, number of release sites, and quantal size. Existing methods of estimating the quantal parameters based on multiple probability fluctuation analysis (MPFA) are limited by their requirement for long recordings to acquire substantial data sets. We therefore devised an algorithm, termed Bayesian Quantal Analysis (BQA), that can yield accurate estimates of the quantal parameters from data sets of as small a size as 60 observations for each of only 2 conditions of release probability. Computer simulations are used to compare its performance in accuracy with that of MPFA, while varying the number of observations and the simulated range in release probability. We challenge BQA with realistic complexities characteristic of complex synapses, such as increases in the intra- or intersite variances, and heterogeneity in release probabilities. Finally, we validate the method using experimental data obtained from electrophysiological recordings to show that the effect of an antagonist on postsynaptic receptors is correctly characterized by BQA by a specific reduction in the estimates of quantal size. Since BQA routinely yields reliable estimates of the quantal parameters from small data sets, it is ideally suited to identify the locus of synaptic plasticity for experiments in which repeated manipulations of the recording environment are unfeasible.

Despite recent advances in mass spectrometry, proteomic characterization of transport vesicles remains challenging. Here, we describe a multivariate proteomics approach to analyzing clathrin-coated vesicles (CCVs) from HeLa cells. siRNA knockdown of coat components and different fractionation protocols were used to obtain modified coated vesicle-enriched fractions, which were compared by stable isotope labeling of amino acids in cell culture (SILAC)-based quantitative mass spectrometry. 10 datasets were combined through principal component analysis into a "profiling" cluster analysis. Overall, 136 CCV-associated proteins were predicted, including 36 new proteins. The method identified >93% of established CCV coat proteins and assigned >91% correctly to intracellular or endocytic CCVs. Furthermore, the profiling analysis extends to less well characterized types of coated vesicles, and we identify and characterize the first AP-4 accessory protein, which we have named tepsin. Finally, our data explain how sequestration of TACC3 in cytosolic clathrin cages causes the severe mitotic defects observed in auxilin-depleted cells. The profiling approach can be adapted to address related cell and systems biological questions.

The fast inhibitory neurotransmitters glycine and GABA are co-localized in synaptic terminals of inhibitory interneurons in the spinal cord and co-released onto lumbar motoneurons in neonatal rats. We performed whole-cell voltage-clamp experiments on spinal cord preparations obtained from juvenile (P8-14) mice to determine whether inhibitory currents exhibited GABAergic components in motoneurons of animals of weight-bearing age. Subsequently we established whether or not GABA is co-released at glycinergic synapses onto motoneurons by determining if it conferred modulatory effects on the kinetics of glycinergic currents. Exponential fitting analysis showed that evoked and miniature inhibitory post-synaptic currents (IPSCs) were best-fitted with a single decay time constant. Responses recorded from connected interneuron-motoneuron pairs showed no effect of a benzodiazepine or a GABA(A) receptor antagonist. Similarly IPSCs evoked by extracellular stimulation and miniature IPSCs were not affected by either agent, indicating the absence of co-detection. Experimental manipulation of the relative content of pre-synaptic GABA and glycine conferred no effect on post-synaptic responses. It is thus unlikely that GABA is co-released in biologically relevant amounts at glycinergic synapses onto lumbar motoneurons in mice of this age.

Reading the spike coding of hypothalamic neurones presents a considerable challenge because they exhibit highly irregular firing patterns. Electrophysiologists working in the motor and sensory systems, in which neurones fire more regularly, have devised satisfactory methods to describe the firing of cells, although the statistical assumptions that underlie the methods do not apply to hypothalamic neurones. Measurement of neural activity is nevertheless vital to characterise the activity of neuroendocrine cells. It has thus become necessary to develop methods suitable for the analysis of the highly irregular spike discharge patterns of both spontaneous and stimulus-evoked firing of hypothalamic neurones. We review techniques used to meet this challenge and demonstrate their considerable capacity to address important physiological questions. We also introduce a novel approach for valid statistical estimation of the information conveyed by the response of a single neurone to a periodic stimulus. The approach demonstrated significant diurnal rhythms of synaptic connectivity between hypothalamic nuclei., (© 2010 The Authors. Journal of Neuroendocrinology © 2010 Blackwell Publishing Ltd.)

Novel measures of coding based on interspike intervals were used to characterise the rhythms of single unit activity in the supraoptic nucleus during the day/night cycle in urethane-anaesthetised rats in vivo. Both continuously firing and phasic cells showed significant (P < 0.001) diurnal rhythms of spike frequency and in the irregularity of firing, as quantified by the log interval entropy (ENT). Comparison of rhythms in log interval ENT showed that the amplitude of the rhythms was greater for the continuously firing cells than for the phasic cells (P = 0.002). Rhythms persisted after hypertonic stimulation or pinealectomy and both treatments reduced the amplitude significantly only for the continuously firing cell group. By contrast, the mesor (i.e. mid-point of the rhythm) was reduced only for the phasic cell group. A similar analysis applied to the activity of cells of the suprachiasmatic nucleus showed that, after pinealectomy, there was a significant rhythm in ENT (P < 0.001) but not firing rate; however, the amplitude of the rhythm in ENT was attenuated (P = 0.047). Diurnal changes in the electrical activity of supraoptic cells are consistent with previously reported circadian changes in magnocellular neuropeptide release. Differences between continuous and phasic cell groups in the effects of osmotic stimulation on rhythmic activity indicate that the two cell types differ in their coding of osmolality and zeitgeber time information. The different effects of pinealectomy on the supraoptic and suprachiasmatic nuclei suggest that removal of endogenous melatonin unmasks a difference in circadian coding between the two nuclei.

The mammalian circadian pacemaker, the suprachiasmatic nucleus (SCN), contains receptors to the adipose tissue hormone leptin. In the present study, the effects of leptin on the electrophysiological activity of the SCN cells were characterised in vitro in rat brain slices. During extracellular recording, application of 20 nm leptin (n = 36) decreased mean spike frequency (Wilcoxon signed rank test, z = -3.390, P < 0.001) and increased the irregularity of firing measured by the entropy of the log interspike interval distribution (Student's paired t-test, t = 2.377, P = 0.023), but had no consistent effect on spike patterning as measured by the mutual information between adjacent log interspike intervals (z = 0.745, P = 0.456). Intracellular current-clamp recordings (n = 25) revealed a hyperpolarising effect of 20 nm leptin on SCN neurones (z = -2.290, P = 0.022). The hyperpolarisation largely resulted from the effect of leptin on the subgroup of cells (n = 13) that generated 'rebound' spikes upon termination of a hyperpolarising current pulse (z = -2.697, P = 0.007). Leptin application also increased the group mean duration of the afterhyperpolarisation (n = 25, t = 2.512, P = 0.023). The effects of leptin on extracellularly recorded spike activity were consistent with the changes in membrane potential and spike shape. They suggest that leptin can directly modulate the electrical properties of SCN neurones and, in this way, contribute to the mechanism by which metabolic processes influence the circadian clock.

Neural information is conveyed by action potentials along axons to downstream synaptic targets. Synapses permit functionally relevant modulation of the information transmitted by converging inputs. Previous studies have measured the amount of information associated with a given stimulus based either on spike counts or on the relative frequencies of spike sequences represented as binary strings. Here we apply information theory to the phase-interval stimulus histogram (PhISH) to measure the extent of the stimulus-evoked response using the statistical relationship between each interspike interval and its phase within the stimulus cycle. We used the PhISH as a novel approach to investigate how different osmotic states affect the flow of information through the osmoreceptor complex of the hypothalamus. The amount of information conveyed from one (afferent) element of the complex, the anteroventral region of the third ventricle (AV3V), to another (an efferent element), the supraoptic nucleus, was increased by hypertonic stimulation (intravenous mannitol, z = 4.39, P < 0.001) and decreased by hypotonic stimulation (intragastric water, z = -3.37, P < 0.001). Supraoptic responses to AV3V stimulation differed from those that follow stimulation of a hypothalamic element outside the osmoreceptor complex, the suprachiasmatic nucleus (SCN), which also projects to the supraoptic nucleus. Thus osmosensitive gain control mechanisms differentially modulate osmotically dependent and osmotically independent inputs, and enhance the osmoresponsiveness of supraoptic cells within a physiological range. The value of the novel approach is that its use is not limited to the osmoreceptor ensemble but it can be used to investigate the flow of information throughout the central nervous system.

The effects of the application of melatonin in vitro on the electrophysiological activity of suprachiasmatic neurones were characterised using novel measures of coding based on the analysis of interspike intervals. Perfusion of 1 nM melatonin in vitro (n = 53) had no consistent effect on mean spike frequency (Wilcoxon's sign rank, z = -0.01, P = 0.989), but increased the irregularity of firing (Student's paired t-test, t = -3.02, P = 0.004), as measured by the log interval entropy, and spike patterning (z = -3.43, P < 0.001), as measured by the mutual information between adjacent log intervals. Intracellular recordings in vitro in current clamp mode showed that 1 nM melatonin significantly hyperpolarised (n = 11, z = -2.35, P = 0.019) those cells that showed 'rebound' spikes upon termination of a hyperpolarising current pulse. Grouping all cells together (n = 27), melatonin application decreased the duration of the afterhyperpolarisation (z = -2.49, P = 0.013) and increased the amplitude of the depolarising afterpotential (z = -2.71, P = 0.007). The effects of melatonin seen in vitro from extracellular recordings on interspike interval coding were consistent with the changes in spike shape seen from intracellular recordings. A melatonin-induced increase in the size of the depolarising afterpotential of suprachiasmatic cells might underlie the increased irregularity of spike firing seen during the subjective night time. The method of analysis demonstrated a difference in spike firing that is not revealed by frequency alone and is consistent with the presence of a melatonin-induced depolarising current.

Novel measures of coding based on interspike intervals were used to characterize the responses of supraoptic cells to osmotic stimulation. Infusion of hypertonic NaCl in vivo increased the firing rate of continuous (putative oxytocin) cells (Wilcoxon z= 3.84, P= 0.001) and phasic (putative vasopressin) cells (z= 2.14, P= 0.032). The irregularity of activity, quantified by the log interval entropy, was decreased for continuous (Student's t= 3.06, P= 0.003) but not phasic cells (t= 1.34, P= 0.181). For continuous cells, the increase in frequency and decrease in entropy was significantly greater (t= 2.61, P= 0.036 and t= 3.06, P= 0.007, respectively) than for phasic cells. Spike patterning, quantified using the mutual information between intervals, was decreased for phasic (z=-2.64, P= 0.008) but not continuous cells (z=-1.14, P= 0.256). Although continuous cells showed similar osmotic responses to mannitol infusion, phasic cells showed differences: spike frequency decreased (z=-3.70, P < 0.001) and entropy increased (t=-3.41, P < 0.001). Considering both cell types together, osmotic stimulation in vitro using 40 mm NaCl had little effect on firing rate (z=-0.319, P= 0.750), but increased both entropy (t= 2.75, P= 0.010) and mutual information (z=-2.73, P= 0.006) in contrast to the decreases (t= 2.92, P= 0.004 and z=-2.40, P= 0.017) seen in vivo. Responses to less severe osmotic stimulation with NaCl or mannitol were not significant. Potassium-induced depolarization in vitro increased firing rate (r= 0.195, P= 0.034), but the correlation with decreased entropy was not significant (r=-0.097, P= 0.412). Intracellular recordings showed a small depolarization and decrease in input resistance during osmotic stimulation with NaCl or mannitol, and membrane depolarization following addition of potassium. Differences in responses of oxytocin and vasopressin cells in vivo, suggest differences in the balance between the synaptic and membrane properties involved in coding their osmotic responses. The osmotic responses in vivo constrasted with those seen in vitro, which suggests that, in vivo, they depend on extrinsic circuitry. Differences in responses to osmolality and direct depolarization in vitro indicate that the mechanism of osmoresponsiveness within a physiological range is unlikely to be fully explained by depolarization.

In this paper, we compare existing methods for quantifying the coding capacity of a spike train, and review recent developments in the application of information theory to neural coding. We present novel methods for characterising single-unit activity based on the perspective of a downstream neurone and propose a simple yet universally applicable framework to characterise the order of complexity of neural coding by single units. We establish four orders of complexity in the capacity for neural coding. First-order coding, quantified by firing rates, is conveyed by frequencies and is thus entirely described by first moment processes. Second-order coding, represented by the variability of interspike intervals, is quantified by the log interval entropy. Third-order coding is the result of spike motifs that associate adjacent inter-spike intervals beyond chance levels; it is described by the joint interval histogram, and is measured by the mutual information between adjacent log intervals. Finally, nonstationarities in activity represent coding of the fourth-order that arise from the effects of a known or unknown stimulus.

The suprachiasmatic nucleus is regarded as the main mammalian circadian pacemaker but evidence for rhythmic firing of single units in vivo has been obtained only recently. The present study was undertaken to determine if rhythms could be seen using measures of activity in addition to the mean spike frequency. We investigated whether there were changes in the irregularity of cell activity measured by the disorder of the interspike interval distribution for neurones recorded in vivo and in vitro. By plotting the entropy of the log interval histogram that quantifies the coding capacity for each action potential against the respective zeitgeber time, we describe oscillations of spike activity in vivo. Entropy measures have the advantage over variances in that they quantify aspects of the shape of the distribution and not just the dispersion. One hundred and sixty-six cell recordings from the suprachiasmatic nucleus showed a significant rhythm in entropy with an oscillatory trend in the data (P < 0.001) showing a trough towards the end of the light period and a peak in the mid-dark period. There was a similar rhythm for the cells recorded from the peripheral zone (n = 209, P = 0.037). In separate experiments in vitro, to investigate the relationship between mean spike frequency and entropy, potassium-induced depolarization of cells recorded during the subjective night was correlated with a significant increase in mean spike frequency (r = 0.259, P = 0.011) and a decrease in entropy (r = -0.296, P = 0.004). The negative correlation between the entropy and mean spike frequency of cells recorded in vitro was significantly different from that seen in vivo (F = 15.5, P < 0.001), which may reflect differences in the balance between deterministic and stochastic influences on spike occurrence. The study shows that while there is a rhythm of mean spike frequency, parameters based on the variability of interspike interval distributions also display rhythmic changes over the day-night cycle.

Novel approaches to the characterization of coding carried by spike trains are discussed. Measuring firing frequency alone may only partially reflect spike patterning, and can only quantify changes of the most obvious kind. We have devised a method that combines probabilistic and information approaches to quantify the variability of the interspike intervals in a way that is independent of spike frequency. To illustrate the technique, the firing of an oxytocin cell and a vasopressin cell were compared before and after osmotic stimulation. A bimodal lognormal function was fitted to the interspike interval histograms. The entropy of the log interval histogram was used to measure the variability of intervals and to reflect the coding capacity of the cell per spike. A perfect metronome shows no variability in interval and thus has no greater coding capacity than is conveyed by its frequency, whereas the variability of intervals of magnocellular neurones means that their irregular activity has greater potential for coding. While the mean spike frequency increased in both the oxytocin and vasopressin cells in response to osmotic stimulation, the changes in their irregularity showed differences. Osmotic stimulation reduced the entropy of the oxytocin cell, reflecting an increase in the regularity of its spike activity. Conversely, osmotic stimulation had little effect on the entropy of the vasopressin cell. Such differences are not evident from a simple inspection of ratemeter activity. The comparison highlights the limitations of mean spike frequency as a measure of spike coding. Parameters based on the interspike intervals constitute informative measures of spike activity that allow objective comparisons to be made between the activity under different physiological conditions.

Measuring spike coding objectively is essential to establish whether activity recorded under one set of conditions is truly different from that recorded under another set of conditions. However, there is no generally accepted method for making such comparisons. Measuring firing frequency alone only partially reflects spike patterning. In this paper, novel quantities based on the logarithmic interspike intervals are proposed as useful measures of spontaneous activity. We illustrate the methods by comparing extracellular recordings from magnocellular cells of the rat supraoptic nucleus in vivo and in vitro and between oxytocin and vasopressin cells in vivo. A bimodal Gaussian function fitted to the log interspike interval histogram accurately described the distribution profile for very different types of activity. We introduce the entropy of the log interval distribution as a novel quantity that measures the capacity of a cell to encode information other than a constant instantaneous frequency. Unlike existing entropy measures that are based on spike counts, it quantifies the variability in the interval distribution. In addition, the mutual information between adjacent log intervals is proposed as an objective measure of patterned activity. For cells recorded in vivo and in vitro, there was no significant difference in mean spike frequencies but there were differences in the log interval entropy (t = -4.97, P < 0.001) and the mutual information (z = -2.64, P < 0.01). The differences may result from the disruption of connections in the slice preparation. When a comparison was made between the spike activity of oxytocin and vasopressin cells recorded in vivo, there was a difference in mutual information (z = 5.15, P < 0.001) but not in mean spike frequency. Both comparisons highlight the potential limitations of using mean spike frequency alone as a measure of spike coding. We propose that our novel parameters based on interval analysis constitute informative measures of spontaneous activity under different physiological conditions.