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In vivo, most vasopressin cells of the hypothalamic supraoptic nucleus fire action potentials in a ‘phasic’ pattern when the systemic osmotic pressure is elevated, while most oxytocin cells fire continuously. The phasic firing pattern is believed to arise as a consequence of intrinsic activity-dependent changes in membrane potential, and these have been extensively studied in vitro. Here we analysed the discharge patterning of supraoptic nucleus neurones in vivo, to infer the characteristics of the post-spike sequence of hyperpolarization and depolarization from the observed spike patterning. We then compared patterning in phasic cells in vivo and in vitro, and we found systematic differences in the interspike interval distributions, and in other statistical parameters that characterized activity patterns within bursts. Analysis of hazard functions (probability of spike initiation as a function of time since the preceding spike) revealed that phasic firing in vitro appears consistent with a regenerative process arising from a relatively slow, late depolarizing afterpotential that approaches or exceeds spike threshold. By contrast, in vivo activity appears to be dominated by stochastic rather than deterministic mechanisms, and appears consistent with a relatively early and fast depolarizing afterpotential that modulates the probability that random synaptic input exceeds spike threshold. Despite superficial similarities in the phasic firing patterns observed in vivo and in vitro, there are thus fundamental differences in the underlying mechanisms.
In vivo, most magnocellular vasopressin cells of the hypothalamic supraoptic and paraventricular nuclei fire action potentials (spikes) in a distinctive ‘phasic’ pattern when the systemic osmotic pressure is elevated. Phasic firing comprises alternating periods of relatively stable activity and electrical silence, and this pattern optimizes the efficiency of stimulus–secretion coupling at the neurosecretory nerve terminals of these cells in the neurohypophysis (see Bourque & Renaud, 1990; Leng et al. 1999 for reviews). However, not all vasopressin cells fire phasically in vivo. Even after osmotic stimulation, some fire continuously, in a manner superficially like that of oxytocin cells (see Brimble & Dyball, 1977). It is not clear whether these non-phasic ‘vasopressin’ cells lack the intrinsic mechanisms that underlie phasic firing, or whether they are oxytocin cells misidentified as vasopressin cells through imprecision in functional identification.
Phasic firing has been studied extensively in in vitro preparations, and studies using intracellular or patch-clamp recording have provided much of our current understanding of how this activity is generated. In vitro, in many supraoptic neurones, spikes are followed by a fast hyperpolarizing afterpotential (HAP) (Bourque et al. 1985) and a superimposed, slower afterhyperpolarization (AHP) (Armstrong et al. 1994) that reflect activation of voltage- and Ca2+-dependent K+ conductances. The net hyperpolarization is followed in turn by a depolarization, reflecting further superposition of a slow Ca2+-dependent depolarizing afterpotential (DAP) (Andrew & Dudek, 1983). Phasic bursts appear to arise through summation of DAPs to form a plateau potential, and spikes generated from this plateau appear to arise regeneratively, as subsequent DAPs approach spike threshold independently of synaptic input (Andrew & Dudek, 1983; Roper et al. 2003). Whether synaptic input in the form of excitatory postsynaptic potentials (EPSPs) is necessary for phasic firing in vitro is unclear; phasic firing has been observed to persist after pharmacological blockade of synaptic transmission (Hatton, 1982), but, in vivo, phasic firing is inhibited by glutamate antagonists (Nissen et al. 1995).
All in vitro preparations of the supraoptic nucleus entail extensive deafferentation, for there is little or no direct synaptic connectivity between supraoptic neurones. The major sources of input are the brainstem, structures adjacent to the lamina terminalis of the third ventricle, and the immediate perinuclear zone (Hatton, 1990; Cunningham & Sawchenko, 1991; Armstrong, 1995). Some perinuclear projections are intact in slice preparations; more are intact in explant preparations, which also encompass the organum vasculosum of the lamina terminalis (OVLT). However, it seems surprising that the discharge patterning of vasopressin cells should be similar in vitro as in vivo despite deafferentation. This persuaded us to look more closely at phasic firing in vivo and in vitro.
We analysed supraoptic neurones in vivo, comparing activity in oxytocin cells with that observed in phasic cells within bursts, and in non-phasic ‘vasopressin’ cells. The functional significance of the HAP, AHP and DAP is thought to lie in their effects on spike patterning, and here we sought to work in the inverse direction – to infer the characteristics of the post-spike sequence of hyperpolarization and depolarization from the observed spike patterning. We then compared patterning in phasic cells in vivo and in vitro.
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Studies in vitro have led to the hypothesis that phasic firing is a regenerative process resulting from large, slow, post-spike DAPs that follow a post-spike hyperpolarization mediated by a fast HAP and slower AHP. This conclusion is most fully expressed in a recent computational modelling study (Roper et al. 2003, 2004) based on in vitro data (Teruyama & Armstrong, 2002), which simulates phasic firing by a fully regenerative mechanism, via a calcium- and voltage-dependent DAP with a time-to-peak of ∼300 ms. This slow time-to-peak is generally consistent with direct observations of the DAP in vitro, thus for instance, Li & Hatton (1997), with patch-clamp recordings from supraoptic neurones in horizontal slices of the rat hypothalamus, reported that DAPs following single evoked spikes had a time-to-peak of 320 ms.
This hypothesis leads to a number of predictions about spike patterning within bursts. If spikes within bursts arise as a result of a predominantly deterministic process (i.e. a regenerative mechanism), then spike timing should be far from random. In this study, we used statistical approaches to test this prediction.
If spike timing within bursts results mainly from a random Poissonian process, then we would expect: (1) the interspike interval distributions should be well fitted by a single negative exponential, with a coefficient of variation close to 100%; (2) the hazard should be independent of time after a spike; and (3) there should be no significant correlation between adjacent intervals within a burst. Conversely, for spikes arriving as a result of slow DAPs following a HAP/AHP, we would expect: (1) the interval distributions should be distributed more symmetrically about a mean that reflects the timing of the DAP; with a relatively low coefficient of variation; (2) the hazard functions should reflect the dynamics of the sequence of hyperpolarization and depolarization and in particular, should show a maximum at the expected time that the DAP reaches spike threshold; (3) at increasingly depolarized resting potentials the DAP should reach spike threshold sooner after a spike, so the mode of the interspike interval distribution and the peak of the hazard function should be inversely related to average firing rate; and (4) adjacent intervals should be correlated, reflecting the long-lasting effects of summation of slow DAPs.
By these criteria, the present analysis of recordings in vitro supports the hypothesis that spikes within phasic bursts in vitro result from a mainly deterministic process. In vitro, spikes in phasic cells are generated at long, relatively regular intervals. The interval distributions are very poorly fitted by negative exponentials. The shape of the hazard functions suggests that maximum excitability is reached several hundred milliseconds after a spike. The coefficient of variation is typically well below 100, and there are relatively strong positive correlations between adjacent interspike intervals. These observations suggest that spikes in phasic bursts in vitro arise by a regenerative mechanism that involves a slow or late DAP that reaches spike threshold, or which falls just short of threshold.
However, analysis of phasic firing in vivo results in very different conclusions. Interspike interval distributions and hazard functions indicate a sequence of post-spike refractoriness followed by hyperexcitability as expected from a sequence of HAP/DAP, but the dynamics of these changes in excitability are very different to in vitro observations. The post-spike hyperexcitability is early, small, and transient compared with in vitro observations. Most of the tail of the interspike interval distribution is well fitted by a single negative exponential, and firing within bursts in vivo is relatively irregular, with a coefficient of variation close to 100%, suggesting that most spikes arise from a random process. Consistent with the dominance of stochastic processes, order effects are very weak: the length of any particular interval is virtually independent of the length of the preceding interval, implying that post-spike changes in excitability within bursts are of short duration.
A previous statistical analysis of phasic firing in vivo drew similar conclusions to the present study: Poulain et al. (1988), in an analysis of phasic firing in supraoptic neurones from lactating rats, concluded that activity within bursts is close to what would be expected of a random process subject to a short refractory period (see Fig. 20 of Poulain et al. 1988). A recent comparison of the statistical discharge of continuously firing supraoptic neurones in vivo and in vitro, independently of the present study, also concluded that discharge is much more regular in vitro than in vivo (Bhumbra & Dyball, 2004).
Here, as in many previous studies (e.g. Hatton, 1982; Mason, 1983b; Andrew & Dudek, 1984; Inenaga et al. 1992, 1993), supraoptic neurones in hypothalamic slices in vitro were recorded in 5 mm KCl (total [K+] 6.25 mm), a condition expected to lead to a modest, sustained subthreshold depolarization. Rather than depolarize cells by raising extracellular [K+], Haller et al. (1978) and Haller & Wakerley (1980) used bath application of glutamate to observe phasic firing in hypothalamic slices. These papers display only five examples of interspike interval distributions from phasic cells, but each of these is relatively symmetrical, with modes between 80 and 130 ms, consistent with the in vitro recordings described here, but different from the in vivo recordings described here. We also analysed seven phasic cells recorded from explant preparations which better preserve the dendritic trees of supraoptic neurones, and which retain more afferent input than slices. In these preparations spontaneous phasic firing can be observed without recourse to extrinsic depolarization. These neurones all showed a late peak in post-spike excitability like phasic neurones from slices, but firing within bursts, though more regular than activity in vivo, was less regular than activity within bursts in slices.
The present in vivo recordings, like those of Poulain et al. (1988), and indeed, like virtually all studies of supraoptic neurones in vivo, were mainly made under urethane anaesthesia. Urethane is the anaesthetic of choice for the magnocellular system since the physiological reflexes – in particular the suckling-induced milk-ejection reflex are intact under urethane, but impaired by most anaesthetics. The advantage of urethane as an anaesthetic is that it ‘produces a long-lasting steady level of surgical anaesthesia, and has minimal effects on autonomic and cardiovascular systems’ (Hara & Harris, 2002). Urethane appears to exert its anaesthetic action by a modest effect on many channels rather than a dominant action on any one: at anaesthetic doses, urethane modestly potentiates the actions of GABA, glycine and acetylcholine, and modestly depresses AMPA and NMDA actions (Hara & Harris, 2002). By contrast, the anaesthetic action of pentobarbitone is thought to result from a large and relatively selective potentiation of GABA actions. However, the structural characteristics of phasic firing do not much differ between urethane or pentobarbitone anaesthesia, nor do they differ between virgin, pregnant and lactating rats, nor do they differ between rat strains, nor do they differ between dorsal and ventral surgical approaches. The consistent differences are between in vivo preparations and in vitro preparations.
The simplest description of the observed differences is that in vitro, both the HAP and the DAP are larger and slower than in vivo. What might account for this? Since both the HAP and the DAP are Ca2+-regulated processes, altered Ca2+ buffering seems a possibility, as Ca2+ buffering is critical for patterning of activity of supraoptic neurones in vitro (Li et al. 1995). However, a second possibility is that loss of tonic synaptic input alters the passive electrical properties of supraoptic neurones. There have been only two brief reports of intracellular recordings from supraoptic neurones in vivo (Bourque & Renaud, 1991; Dyball et al. 1991), but both confirm that there is a much higher PSP frequency in vivo, and a lower input resistance. Recently, Destexhe et al. (2003) compared the electrophysiological properties of neocortical neurones in vivo and in vitro; like the present study, firing in vivo was much more irregular than in vitro, and these authors used computational modelling to study the differences in patterning. Intracellular recordings indicated a massively lower cell input resistance in vivo, as expected from a much greater afferent input, and computational modelling showed that this ‘high-conductance’ state of neurones in vivo was accompanied by an increase in voltage attenuation, and a reduction in membrane time constant compared with the in vitro state. These observations indicate a possible explanation for the apparent differences in DAP amplitude and dynamics inferred from the present study. The duration of the DAP far exceeds the membrane time constant, but the differences in hazard functions between in vivo and in vitro preparations indicate differences in the activation time of the DAP or inactivation time of post-spike hyperpolarization, and both of these may be expected to be influenced by membrane time constant.
Here, we have also shown that non-phasic cells in the supraoptic nucleus that are functionally identified as vasopressin cells show discharge patterning similar to the intraburst activity of phasic cells, and dissimilar to that of oxytocin cells. In non-dehydrated rats, not all non-phasic cells are oxytocin cells (typically fewer than half of all vasopressin cells fire phasically), so other criteria are needed, particularly to identify cells in non-lactating rats. Oxytocin release is increased following CCK injection, and CCK consistently activates oxytocin cells identified by reflex milk ejection; conversely, vasopressin release is inhibited by CCK (Verbalis et al. 1986a,b) and CCK inhibits some non-phasic cells. Increases in blood pressure induced for instance by phenylephrine inhibit phasic cells and some non-phasic cells, but rarely inhibit identified oxytocin cells (Leng et al. 1991). Thus non-phasic cells that are inhibited by CCK are probably mostly vasopressin cells. Spikes in phasic cells and non-phasic ‘vasopressin’ cells are followed by a transient refractoriness consistent with a HAP/AHP, succeeded by a fast, transient hyperexcitability consistent with a DAP, though the inferred DAP is more transient for non-phasic vasopressin cells than for phasic cells. Oxytocin cells in vivo showed post-spike refractoriness consistent with a HAP/AHP but no indications of a post-spike hyperexcitability as would be expected from the effects of a DAP.
Thus vasopressin cells in vivo consistently differ from oxytocin cells in displaying a transient post-spike hyperexcitability, indicative of a fast DAP, but this is not always associated with phasic firing. The hyperexcitability lasts for long enough to summate with repeated spiking, though such order effects are weak. In non-phasic vasopressin cells the post-spike hyperexcitability was more transient than in phasic cells, with potentially less opportunity to summate. Thus the conclusion that phasic firing reflects spike activity above a plateau resting potential that is sustained by summation of DAPs appears plausible. However, while this general conclusion originally derived from in vitro studies seems valid in vivo, the suggestion that phasic firing is regenerative and independent of synaptic input does not seem tenable as a description of phasic firing in vivo. Indeed, a recent study shows that evoked phasic bursts are not regenerative in vivo, but require continued excitatory input even at the onset of bursts (Brown et al. 2004).
The present type of analysis cannot address the issue of what terminates bursts – and the termination of bursts and the long refractoriness of burst generation establish the phasic firing pattern in conjunction with the mechanisms that sustain firing within bursts. The mechanisms underlying burst generation remain incompletely understood. Recently, it has become apparent that activity-dependent dendritic secretion of vasopressin (Ludwig & Leng, 1997) and dynorphin, coexpressed with vasopressin, may have an autoregulatory role in burst patterning (Brown et al. 1998, 2004) along with activity-dependent generation of nitric oxide (Srisawat et al. 2000). Recently, Brown & Bourque (2004) showed that burst termination results from activity-dependent inhibition of plateau potentials by endogenous dynorphin, and Roper et al. (2004) proposed a model of burst generation and termination in which dynorphin secretion from dendrites terminates bursts by a slow progressive desensitization to calcium of the plateau potential. Oxytocin cells also coexpress opioid peptides, and synthesize nitric oxide, though at lower levels than vasopressin cells (see Brown et al. 2000; Srisawat et al. 2000). Thus some basic mechanisms that are important for phasic firing are present in both cell types.
In summary, phasic firing in vasopressin cells in vitro appears to arise regeneratively from a relatively slow, late DAP that approaches or exceeds spike threshold. By contrast, in vivo activity is dominated by stochastic rather than deterministic mechanisms, and appears to reflect a relatively early and fast DAP that peaks below threshold but which modulates the probability that random synaptic input exceeds spike threshold. Recent work indicates that computational modelling may have the power to reconcile observations made in vitro with observations made in vivo.