differences in spike train variability in rat vasopressin and oxytocin neurons and their...

J Physiol 581.1 (2007) pp 221–240 221

Differences in spike train variability in rat vasopressin andoxytocin neurons and their relationship to synaptic activity

Chunyan Li, Pradeep K. Tripathi and William E. Armstrong

Department of Anatomy and Neurobiology, University of Tennessee Health Science Center, Memphis, TN 38163, USA

The firing pattern of magnocellular neurosecretory neurons is intimately related to hormone

release, but the relative contribution of synaptic versus intrinsic factors to the temporal dispersion

of spikes is unknown. In the present study, we examined the firing patterns of vasopressin (VP)

and oxytocin (OT) supraoptic neurons in coronal slices from virgin female rats, with and without

blockade of inhibitory and excitatory synaptic currents. Inhibitory postsynaptic currents (IPSCs)

were twice as prevalent as their excitatory counterparts (EPSCs), and both were more prevalent

in OT compared with VP neurons. Oxytocin neurons fired more slowly and irregularly than VP

neurons near threshold. Blockade of Cl− currents (including tonic and synaptic currents) with

picrotoxin reduced interspike interval (ISI) variability of continuously firing OT and VP neurons

without altering input resistance or firing rate. Blockade of EPSCs did not affect firing pattern.

Phasic bursting neurons (putative VP neurons) were inconsistently affected by broad synaptic

blockade, suggesting that intrinsic factors may dominate the ISI distribution during this mode in

the slice. Specific blockade of synaptic IPSCs with gabazine also reduced ISI variability, but only

in OT neurons. In all cases, the effect of inhibitory blockade on firing pattern was independent

of any consistent change in input resistance or firing rate. Since the great majority of IPSCs are

randomly distributed, miniature events (mIPSCs) in the coronal slice, these findings imply that

even mIPSCs can impart irregularity to the firing pattern of OT neurons in particular, and could

be important in regulating spike patterning in vivo. For example, the increased firing variability

that precedes bursting in OT neurons during lactation could be related to significant changes in

synaptic activity.

(Received 27 October 2006; accepted after revision 26 February 2007; first published online 1 March 2007)

Corresponding author W. E. Armstrong: Department of Anatomy and Neurobiology, University of Tennessee Health

Science Center, Memphis, TN 38163, USA. Email: [email protected]

Magnocellular neurosecretory cells (MNCs) in thesupraoptic nucleus (SON) and paraventricular nucleus(PVN) are specialized to synthesize the neurohormonesvasopressin (VP) and oxytocin (OT). The axons of theseneurons transport the secretory vesicles from the soma tothe neurohypophysis, where the hormones are releasedinto the blood circulation. Studies in vivo (Poulain &Wakerley, 1982) and in vitro (Cazalis et al. 1985; Bicknell,1988) have demonstrated a close correlation betweenthe electrophysiological activity of these neurons and thehormone release at the neurohypophysis. In both cell types,interspike interval (ISI) irregularity could contribute toenhanced release, especially when short intervals areclustered (Cazalis et al. 1985; Bicknell, 1988; Poulain et al.1988).

In lactating rats, OT neurons show highly stereotyped,synchronized milk ejection bursts of 50–80 Hz for 1–4 s(Poulain & Wakerley, 1982). This short burst is super-

This paper has supplemental material.

imposed on a slower background of spike dischargeranging from < 1 to ∼10 Hz, and ranging in patternfrom irregular to regular (a.k.a. fast continuous). Recentstudies suggest that the probability of milk ejection burstsis related to background discharge, such that a backgroundfiring rate of 1–3 Hz appears critical, and the degree offiring rate variability is positively correlated with burstprobability (Brown & Moos, 1997; Brown et al. 2000; Mooset al. 2004). In response to other stimuli, in either maleor female rats, OT neurons typically increase their firingrate and adopt a continuous activity pattern, not unlikethat which can be observed between milk ejection bursts.Although OT release during milk injection is dependent onglutamate receptor activation (Parker & Crowley, 1993),and GABAergic activity strongly influences OT neurondischarge patterns (Moos, 1995; Leng et al. 2001), theprecise role of synaptic activity in spike distribution isunknown.

Vasopressin neurons can also fire in three modes,including a slow irregular (< 1 Hz), fast continuous

C© 2007 The Authors. Journal compilation C© 2007 The Physiological Society DOI: 10.1113/jphysiol.2006.123810

222 C. Li and others J Physiol 581.1

(> 1 Hz) and a phasic bursting firing (Poulain &Wakerley, 1982; Poulain et al. 1988). The latter twomodes, especially phasic bursting, are adopted during VPrelease in response to stimuli such as dehydration andhypovolaemia. In contrast to OT neurons, this burstingis not synchronized among cells and is composed ofalternating periods of activity (5–15 Hz) and silence,each lasting 20–40 s. Phasic bursting has been studiedin vitro and is associated with intrinsic membraneproperties such as the calcium-dependent depolarizingspike after-potential and its related plateau potential(Bourque, 1986; Renaud & Bourque, 1991). Nevertheless,in vivo, phasic bursting activity of VP neurons is dependentupon excitatory synaptic transmission (Nissen et al. 1995;Brown et al. 2004), and recent studies suggest that spikepatterning within bursts of phasic neurons differs betweenin vivo and in vitro preparations (Sabatier et al. 2004).

Many in vivo patterns of activity of OT and VPneurons can be observed in hypothalamic slices orthe hypothalamo-neurohypophysial explant in vitro,including phasic, continuous and slow irregular activity(Renaud & Bourque, 1991; Armstrong, 1995). Recentreports even demonstrate milk ejection-like activity inpharmacologically treated slices from lactating rats andmale rats (Wang & Hatton, 2004, 2005) and in organotypiccultures (Jourdain et al. 1998; Israel et al. 2003). Inthe present study, we investigated how spontaneousexcitatory and inhibitory synaptic activity contribute tothe distribution of interspike intervals of identified OTand VP neurons in slices from adult virgin female rats,whether both cell types were equally influenced by thisactivity, and whether excitatory and inhibitory activitywere equally influential. Some of these data have previouslybeen published in abstract form (Li & Armstrong, 2002,2003).

Methods

Hypothalamic slice preparation

Female, virgin Sprague–Dawley rats (150–250 g), takenrandomly throughout the oestrus cycle, were deeplyanaesthetized with sodium pentobarbitone (50 mg kg−1,i.p.) and perfused through the left ventricle with 60 mlof ice-cold, low-Na+ (replacing 125 mm NaCl with210 mm sucrose) artificial cerebrospinal fluid (ACSF),which had been fully oxygenated (95% O2–5% CO2). Therat was decapitated and the brain removed to a slushof the low-Na+ ACSF. Coronal slices (250 μm) werecut through the hypothalamus with a vibrating-blademicrotome (VT 1000S, Leica) with the tissue submergedin this slush, which was also fully oxygenated(95% O2–5% CO2), then placed on a nylon net in aretaining chamber containing oxygenated, normal ACSF.The ACSF composition was as follows (mm): KCl, 2.5;

MgSO4, 1.0; NaH2PO4, 1.25; NaHCO3, 26; d-glucose, 20;ascorbic acid, 0.45; CaCl2, 2.0; and NaCl, 125 (osmolality,290–310 mosmol kg−1; pH 7.3–7.4). After incubation at32–34◦C for 45–60 min, the slices were stored at roomtemperature (22–24◦C) in oxygenated ACSF until used.Recordings were made at 32–34◦C, with a perfusion rateof 1.5–2.0 ml min−1.

Drug application

Picrotoxin (100 μm), a blocker of Cl− channels as foundin GABAA receptors, or the more selective GABAA

receptor antagonist gabazine (10 μm) was selected toblock inhibitory transmission. The glutamate receptorantagonist 6,7-dinitro-quinoxaline-2,3(1H,4H)-dione(DNQX, 10 μm) was used to blocked AMPA/kainatereceptors, and kynurenic acid (2 mm), a broad-spectrumglutamate receptor blocker, was used to block bothN-methyl-d-aspartate (NMDA) and non-NMDAreceptor activity. Tetrodotoxin (TTX; 0.5 μm) was used toblock voltage-gated sodium channels and thus the actionpotentials. All drugs were purchased from Sigma/RBI,and applied to the ACSF perifusate.

Whole-cell electrode recording

Recording pipettes (5–7 M�) were pulled fromthin-walled borosilicate capillary tubing (o.d. = 1.5 mm,i.d. = 1.17 mm, Warner Instrument Corp.) using amodel P-80/PC Flaming-Brown horizontal micro-pipette puller (Sutter Instrument Co.). To reducecapacitative artifacts, beeswax was applied to thepipette shank. The patch solution consisted of (mm):potassium gluconate, 140; KCl, 10; Hepes, 10; Mg-ATP, 4; Na-GTP, 0.3; phosphocreatine, 3.5; EGTA, 0.2(osmolality, ∼285 mosmol kg−1; pH 7.2–7.3). Themeasured liquid junction potential using this solutionwas +10 mV, and the data were corrected for this offset.A second patch solution consisted of (mm): CsCl, 120;Hepes, 30; EGTA, 11; MgCl2, 2; CaCl2, 1.0; and MgATP, 4.Caesium hydroxide was used to adjust pH to 7.3. Thefinal osmolality was around 295 mosmol kg−1. The liquidjunction potential with this high-Cl− pipette solution wasrelatively small (∼3 mV), and thus data were not adjusted.Biocytin (2 mg ml−1) was added to the intracellularsolution in order to label neurons for immunochemicalidentification.

Whole-cell recordings were made using either theMulticlamp 700A or Axopatch 200B amplifier and theDigidata interface (1320A or 1322A; Axon Instruments).Under visual guidance, cells lying at 20–80 μm from theslice surface were patched. A small amount of positivepressure was applied through the pipette as it advancedtoward the neuron. Upon contacting the cell membrane,

C© 2007 The Authors. Journal compilation C© 2007 The Physiological Society

J Physiol 581.1 Influence of spontaneous synaptic activity on spike train variability 223

negative pressure was applied until a seal (> 1 G�) wasachieved. Residual electrode capacitance was then negated.Series resistance ranged from 10 to 20 M� once whole-cellmode was achieved and was monitored periodicallybut not compensated. The synaptic activity recorded involtage-clamp mode was digitized at 20 kHz and filteredat 2 kHz. The voltage output for current-clamp mode wasdigitized at 20 kHz and filtered at 10 kHz.

Data were transferred to the host computer (DellPrecision 330) and stored for further analysis on anApple iMac computer. For each neuron, synaptic activitywas first examined in voltage-clamp mode for 1 minperiods to record inhibitory postsynaptic (IPSCs) andexcitatory postsynaptic currents (EPSCs). These weredistinguished from −50 to −60 mV, where IPSCs wereoutward and EPSCs were inward currents, and verifiedfollowing application of drugs to selectively block onetype or the other. Under current clamp, we then collectedspike trains for 5 min periods. In order to normalizebaseline conditions across neurons and preparations, spiketrains were recorded near spike threshold. If the cellwas spontaneously active at rest, negative DC currentwas used to hyperpolarize it closer to spike threshold.If the cell was silent, positive DC current was appliedto depolarize the neuron near threshold so that firingwas just elicited. Thus firing patterns herein are basedon the minimal activity we could achieve (with at leasta 1 Hz mean rate). As will be shown in the Results,the amount of DC injection did not vary significantlypre- and postdrug application. Following these recordings,I–V curves were obtained from current-clamp tracesto estimate input resistance (Rin) using hyperpolarizingsteps (−10 pA, 400 ms). Magnocellular SON neuronswere confirmed at recording by the presence of transientoutward rectification (Renaud & Bourque, 1991;Armstrong & Stern, 1997) using depolarizing currentsteps (+10 pA, 400 ms), and post hoc with immuno-chemistry (see below). We thus measured synapticactivity, spike trains and Rin before and after drugapplications.

Data analysis

Spike times were retrieved in AxoGraph software (AxonInstruments) by setting a spike amplitude threshold of0 mV in order to acquire a serial stream of interspike inter-vals (ISIs). The ISI data were then analysed in Igor 4.0(Wavemetrics, Lake Oswego, OR, USA) using a customizedsubroutine. For irregularly/continuously firing neurons,only stationary spike trains were analysed. Spike trains withlong-term tendencies to change firing rate as detected bythe elevated tail of the serial correlogram were discarded,or in some cases trimmed to remove areas where ratewas changing, so long as a contiguous, stationary areaaccounted for the majority of the recording time. Thiswas important because long-term trends in firing rate

contributed spurious variability. In addition to the meanfiring rate, we used two scalable indices of variability: thecoefficient of variation (CV) of the ISI and the Fano factorof the firing rate. The CV is the ratio of standard deviationto the mean ISI, whereas the Fano factor is the ratio ofthe variance of the number of spikes generated within a1 s time window to the mean number of spikes in thesame bin period (Koch, 1999). Each index is useful fordetermining changes in spike train variability independentof changes in mean firing rate, but their time windowis different, since CV is based on the absolute ISIs andthe Fano factor is integrated over 1 s. We also plotted theISI histogram, instantaneous firing rate over time, jointinterval histogram and serial correlogram for each periodof activity.

Neurons were classified according to their pattern ofactivity with a membrane potential near spike threshold.For phasic neurons, bursts were identified by an Igorsubroutine according to the following criteria: there wasat least one burst and one silent period in the 5 minrecording; the minimum burst duration and silenceduration (when more than one burst was present) wereboth 3 s; and the minimum mean intraburst firing ratewas ≥ 2 spikes s−1 (Poulain et al. 1988; Sabatier et al.2004). The remaining cells were classified as belongingto a continuously/irregularly firing continuum (Poulainet al. 1988). The joint interval histogram plots each ISIversus the ISI immediately following (ISI+1), and is usefulto appreciate the variability of pairs of ISIs, with moreirregularly firing neurons displaying a more disperseddistribution.

The serial correlogram (Perkel et al. 1967) is the plotof the serial correlation coefficient of interval lengths. Theserial correlation coefficient (ρ) of order j is the ratio of thecorresponding autocovariance (Cj) to the interval variance(σ 2):

ρ j = C j

/σ 2

The autocovariance of interval lengths, of lag j, is definedas the following:

C j = E[(Ti − μ)(Ti+ j − μ)]( j = · · · , −1, 0, 1, · · ·)where E is the expectation operator and Ti is the ith inter-spike interval in a spike train, with mean interval μ. Thecovariance of two features measures their tendency to varytogether and is defined as the average of the products of thedeviation of feature values from their means. Therefore, weused the following formula to calculate Cj , and thereforeρ j :

C j =[ ∑

(Ti − μ)(Ti+ j − μ)

]/(n − j − 1)

where n is sample size (the total number of ISIs).The serial correlogram is useful to appreciate the

order dependence of ISIs and to determine long-term



stationarity. If ISIs are independently distributed, ρ

approaches 0. A large serial correlation coefficient,ρ j = Cj/σ

2, (positive or negative) for any order j suggeststhat a particular spike time is dependent on previous ISIs(e.g. a positive correlation at the first order means thatadjacent ISIs covary in length). A prolonged, elevatedtail of the serial correlogram suggests that long-termchanges in firing patterns, such as a slowly deceleratingor accelerating firing rate over the 5 min recording period.For irregularly/continuously firing neurons, we used thisplot to trim data so that long-term trends in firing rate wereremoved and so that relatively stationary distributionswere examined. Otherwise, slow changes in firing rate overtime produce spuriously large CVs.

Excitatory postsynaptic currents and IPSCs werecaptured with AxoGraph or MiniAnalysis (Synaptosoft,Decatur, GA, USA). The amplitude and frequency ofsynaptic events were calculated both before and after drugtreatment. For experiments to study amplified miniatureIPSCs (mIPSCs) with a high-Cl− pipette solution, 10 μm

DNQX and 40 μm 2-amino-5-phosphonopentanoic acid(AP5) were added to block glutamatergic transmission,and 0.5 μm TTX was added to block spikes. When holdingat −60 mV in voltage clamp, mIPSCs were large inwardcurrents. The total amount of mIPSCs includes bothmonoquantal events whose rise and decay could be fullyobserved and multiple events whose rise or decay couldnot be fully observed but had discernible peaks.

Since ISI distributions ranged from normal to Poisson,a non-parametric paired test (Wilcoxon’s signed ranktest) was used to compare data before and after drugapplication. The non-parametric Mann–Whitney U testwas used to determine differences between groups ofneurons, for example, OT versus VP neurons.

Immunohistochemistry

For identification of recorded neurons, the slice wasfixed in sodium phosphate-buffered (0.15 m; pH 7.4) 4%paraformaldehyde and 0.2% picric acid at 4◦C, and storedfor several days before histology. Accumulated sliceswere rinsed thoroughly in sodium phosphate-bufferedsaline (PBS) containing 0.5% Triton X-100 (TX) madefrom 0.01 m sodium phosphate and 0.015 m NaCl, andincubated in primary antibody for 1 or 2 days at 4◦C.Vasopressin neurons were identified by a polyclonalantibody raised in rabbit against VP-neurophysin(VP-NP, provided by Alan Robinson, retired), diluted1:20 000. Oxytocin neurons were labelled by a mousemonoclonal antibody specific for OT-neurophysin PS36or PS38 (OT-NP, provided by Harold Gainer, NIH),diluted 1:500. Primary antibodies were diluted in PBS-TX,0.04% sodium azide, and could be reused for severalincubations. After incubation in primary antibody, theslices were rinsed in three changes of PBS-TX, then placedinto a cocktail of secondary antibodies (1:200; Jackson

Laboratories, West Grove, PA, USA) containing fluoresceinisothiocyanate (FITC)-conjugated goat antirabbitimmunoglobulin (GAR-FL), Texas Red isothiocyanate-conjugated goat antimouse immunoglobulin (GAM-TR)and, to label the biocytin-filled neuron, avidin-AMCA(7-amino-4-methylcoumarin-3-acetic acid; 1:400; VectorLaboratories, Burlington, CA, USA), for 1 or 2 days at 4◦C.The slices were then rinsed with PBS-TX and mountedwith a solution of glycerol and PBS (1:1) for observationunder a fluorescence microscope (Nikon Optiphot).Photographs were captured with a digital camera(Sensyscam, Photometrics), and IPLabs (Scanalytics,Fairfax, VA, USA) software. Neurons were identified aseither OT or VP neurons only if positive staining wasaccompanied by a negative reaction for the other peptide.Only a small minority of neurons reacted with bothantibodies, in agreement with a previous study showingthat a few SON neurons colocalize OT and VP, as wellas their respective mRNAs, in normal animals (Mezey &Kiss, 1991).

The work is approved by the Institutional Animal Careand Use Committee of University of Tennessee HealthScience Center (protocol no. 283), effective date 1/05/07.

Results

Identification of cell types

We were unable to consistently apply the electro-physiological criteria we developed previously, and whichwere very useful for sharp electrode recordings (Stern& Armstrong, 1995; Teruyama & Armstrong, 2002).These criteria include the presence of sustained outwardrectification (SOR) and a rebound depolarization (RD)present in OT but not VP neurons. At this time, we arenot certain why these characteristics are not consistentlyexhibited with whole-cell recording. A recent study usingperforated patch recordings (avoiding dialysis) in SONneurons found expression of the SOR in a majority ofOT but not VP neurons, using voltage-clamp protocols(Hirasawa et al. 2003). Interestingly, these authors alsonoted that most OT neurons also expressed a stronginward rectification at potentials more hyperpolarized(< −70 mV) to the range (−70 to −40 mV) where SORwas obvious.

We recorded 202 SON neurons in slices from 104 virginrats where immunoidentification was attempted. Of these,89 were identified as VP neurons, 83 as OT neurons (Fig. 1)and 30 were unidentified, yielding a success rate of ∼85%.Another 11 neurons were studied in a pilot study whereinimmunoidentification was not attempted.

Firing pattern of SON neurons

Of 157 SON neurons (135 irregularly/continuously firing;22 phasic) examined, 103 (66%) were spontaneously active



Table 1. Comparison of the current injected into cells betweencontrol and drug treatment

Drug Cell type Icontrol (pA) Idrug (pA) P value

Picrotoxin OT (n = 14) −2.8 ± 7.6 −4.5 ± 6.6 P = 0.13VP (n = 21) −8.6 ± 10.6 −10.4 ± 12.5 P = 0.10

DNQX OT (n = 10) −5.5 ± 11.2 −7.2 ± 15.5 P > 0.99VP (n = 10) −8.7 ± 12.4 −8.7 ± 14.1 P = 0.75

Kynurenic OT (n = 9) −3.9 ± 13.4 −2.2 ± 13.4 P = 0.92acid VP (n = 10) −3.6 ± 13.1 −2.5 ± 12.2 P = 0.42

Values are means ± S.D.

at rest and exhibited a range of firing rates and patternsresembling those observed in vivo: continuous firing,irregular firing and phasic firing (Poulain & Wakerley,1982; Poulain et al. 1988). In order to standardizetesting, positive DC current was applied to silent neuronsto bring them near spike threshold, and for somespontaneously firing neurons we applied negative DCcurrent, if necessary, to bring the membrane potentialclose to threshold. For continuously firing neurons, theamount of current injection needed to keep neuronsat threshold was not significantly different before andafter any drug treatment, in either OT or VP neurons(Table 1). Phasic neurons were not considered in thisparticular analysis owing to their lower numbers. Thusfiring patterns herein are based on the minimal activitywe could achieve near threshold. Figure 2 shows the threemain kinds of firing patterns exhibited by SON neurons:fast continuous, with a Gaussian distribution of ISIs;slow irregular, with a Poisson distribution; and finally thephasic bursting characteristic of VP neurons (Poulain et al.1988).

Joint interval histograms showed that irregularly firingneurons exhibited a dispersed distribution of ISI pairs(Fig. 2Cd), whereas more regularly firing neurons showeda dense, symmetric core about the mean ISI (Fig. 2Ad).Phasic bursting neurons showed a relatively symmetriccore about the mean intraburst ISI in the joint ISIhistogram (Fig. 2Bd), similar to the core in continuouslyfiring neurons (Fig. 2Ad) but less dense because spike pairs

A C

D

B

E F

25 μm

*

*

Figure 1. Immuno-identification of OT andVP neuronsImmunohistochemical identification ofbiocytin-filled neurons. A and D arebiocytin-filled neurons recovered withavidin-AMCA. A–C, OT neuron; D–F, VPneuron. B and E show results for antibody toOT-neurophysin (revealed with goat antimouseTexas Red). C and F show results for anantibody to VP-neurophysin (revealed withgoat antirabbit FITC). ∗ in B and F indicates theinjected double-labelled neuron

were confined to bursts. Phasic neurons also often showedsome dispersed, larger ISIs in the joint ISI histogramassociated with interburst intervals; however, longinterburst intervals (> 3 s) are not shown in Fig. 2Bd. Forirregularly firing neurons, the core was sparse, and thedots representing the adjacent pairs of ISIs were widelydispersed (Fig. 2Cd). For the tests described below, we firstdeal with continuously firing neurons.

Synaptic activity is largely from miniature PSCsand largely GABAergic

Holding at −50 to −60 mV in voltage-clamp mode withthe potassium gluconate pipette solution, both inwardEPSCs and outward IPSCs were visible in all neurons.In each cell tested, picrotoxin (100 μm) blocked theoverwhelming majority of IPSCs, and DNQX (10 μm)virtually all EPSCs (Fig. 3).

Initially, we tested the effect of TTX in 11unidentified SON neurons. Tetrodotoxin slightly butsignificantly reduced the frequency of IPSCs (control,3.30 ± 3.06 Hz; TTX, 2.98 ± 2.76 Hz; P = 0.013) andslightly reduced their amplitude (control, 19.2 ± 3.7 pA;TTX, 16.7 ± 3.6 pA; P = 0.051). Neither the frequency(control, 1.84 ± 1.06 Hz; TTX, 1.72 ± 0.89 Hz; P = 0.879)nor the amplitude of EPSCs (control, −23.3 ± 6.2 pA;TTX, −20.7 ± 5.9 pA; P = 0.062) was affected by TTX.These results suggested that the great majority of IPSCsand EPSCs were from miniature events.

We further examined amplified IPSCs with a high-CsClpipette solution on immunoidentified neurons becauseIPSCs exerted a more influential effect on firing patternsthan that of EPSCs in both OT and VP neurons (see‘Effects of blockade of IPSCs on continuously firingneurons’ section). Inhibitory PSCs were isolated byapplying 40 μm AP5 and 10 μm DNQX, and the recordedand biocytin-filled neurons were identified as OT or VP.Tetrodotoxin did not change the frequency of the IPSCs ofthese pooled SON neurons (n = 26; IPSC frequency beforeTTX, 4.19 ± 3.29 Hz; TTX, 4.35 ± 3.69 Hz; P = 0.587).Moreover, the IPSC frequency of OT or VP neurons was notchanged by TTX (Fig. 4). These data also showed that fewer



IPSCs (∼3 Hz) were detected when using the potassiumgluconate pipette solution compared with the high-CsClpipette solution (∼6 Hz). Thus, it is possible that smallIPSCs were masked by noise and could not be detected withpotassium gluconate solution. Another factor contributingto this large difference in IPSC frequency detected bythe two pipette solutions might be the ratio of OT and

Figure 2. Different firing patterns of SON neuronsAa–d, SON neuron exhibiting fast continuous firing. Aa, 10 s trace from 5 min recording shows the regularityof the firing. Ab, ratemeter from a 5 min recording. Ac, ISI histogram of all ISIs shows a normal distribution.Ad, ISI joint interval histogram. Each dot in this plot represents an adjacent pair of ISIs. The regularity of the firingresults in a tight cluster around the mean ISI. Ba–d, SON neuron exhibiting phasic bursting activity characteristicof VP neurons. Ba, 5 min trace showing 4 bursts with long interburst intervals. Bb, ratemeter. Bc, ISI histogram.The 3 long interburst ISIs are excluded. The ISI histogram reveals a mixed distribution of ISIs with essentially threepeaks. Bd, ISI joint interval histogram. The dots are more dispersed, representing larger irregularity of ISI during thebursts compared with the fast continuous neuron, and also show some clustering. Again, the 3 large interburstintervals are not shown. Ca–d, SON neuron exhibiting slow, irregular firing. Ca, a 3 min trace of irregular firing.Cb, ratemeter from a 5 min recording. Cc, ISI histogram of all ISIs reveals a Poisson distribution. Cd, ISI joint intervalhistogram. Note the dispersed dots, representing a more random distribution of ISIs.

VP neurons recorded in the initial study of unidentifiedneurons, because the high-Cl− data revealed that OTneurons have far more IPSCs (9.1 ± 6.7 Hz) than do VPneurons (1.1 ± 0.7 Hz; P < 0.0002).

Inhibitory PSCs were sometimes clustered such that thepeak of one occurred in the decay of another. To determinewhether the frequency of putative monoquantal IPSCs was



affected by TTX, we differentiated these multiple eventsfrom those characterized by a full, smooth rise and decay(Fig. 4). The frequency of these monoquantal events wasnot affected by TTX for either OT or VP neurons. Thedifference between OT and VP neurons was still striking,such that OT neurons (n = 22; 4.3 ± 2.0 Hz) exhibited afrequency of mIPSCs almost fourfold that of VP neurons(n = 13; 1.2 ± 1.0 Hz; P < 0.0001). Tetrodotoxin did notsignificantly alter the amplitude of monoquantal IPSCsin either OT neurons (n = 18; IPSC amplitude beforeTTX, −150.3 ± 58.4 pA; after TTX, −135.5 ± 57.2 pA;P = 0.071) or VP neurons (n = 13; IPSC amplitude beforeTTX, −196.0 ± 61.4 pA; after TTX, −172.5 ± 63.9 pA;P = 0.157).

To determine the temporal distribution of events,we examined intersynaptic intervals of IPSCs in thepresence of DNQX and AP5 in 12 of the 18 OT neurons,chosen because there were sufficient numbers of IPSCs(> 600 events min−1 recording time) to attempt to fit thedistribution. In 11 of these 12 neurons, the tail of theintersynaptic interval histogram was well fitted to a singleexponential and the serial correlogram was flat (Fig. 5).This is consistent with an essentially random distributionof events, which is also consistent with the relative lack ofeffect of TTX on synaptic frequency and amplitude. Thus,even though IPSCs may cluster (Fig. 4), these bouts are forthe most part randomly distributed. In the one neuron thedistribution of which could not be well fitted, there weremultiple peaks in the intersynaptic interval distribution

Figure 3. Picrotoxin blocked IPSCs and DNQX blocked EPSCsAa–c, SON neuron held at −50 mV in voltage clamp. Aa, control state, showing IPSCs (outward currents) andEPSCs (inward currents). Ab, the same cell exposed to 100 μM picrotoxin, which blocked IPSCs but not EPSCs.Ac, expanded trace, showing an EPSC and IPSC. Ba–c, another SON neuron held at −55 mV. Ba, control state,showing IPSCs and EPSCs. Bb, the same cell exposed to 10 μM DNQX, which blocked EPSCs but not IPSCs.Bc, expanded trace, showing an IPSC and EPSC.

and a small oscillation in the serial correlogram,suggesting some order dependence of synapticevents.

Thus these data confirmed that the synaptic activitiesare primarily miniature events in the coronal slice andconfirmed that spontaneous IPSCs, mainly GABAergic innature, are more common than EPSCs.

Effects of blockade of IPSCs on continuouslyfiring VP and OT neurons

We asked whether IPSCs and EPSCs contributeddifferentially to spike distribution, and whether bothOT and VP neurons were affected. We noted withimmuno-identification that irregularly/continuouslyfiring neurons could be either OT or VP neurons.However, some neurons changed their firing pattern tophasic bursting during drug treatment; these and otherphasic neurons will be dealt with below, and for thefollowing analysis we consider only those neurons thatfired irregularly/continuously in both control and drugperiods. For OT neurons, the CV and Fano factor weresignificantly reduced by the blockade of IPSCs, but themean firing rate and input resistance were unaffected(Fig. 6C). Virtually identical results were obtained for VPneurons, i.e. the CV and Fano factor were significantlyreduced after the application of picrotoxin, with noeffects on mean firing rate or input resistance (Fig. 6D).After applying picrotoxin, the initial peak of the averaged



serial correlogram increased after picrotoxin for bothOT (control, 0.180 ± 0.157; picrotoxin, 0.310 ± 0.177;P = 0.0258) and VP neurons (control, 0.287 ± 0.181;picrotoxin, 0.392 ± 0.160; P = 0.0457). This elevationpersisted for 5–10 spikes in both cell types, suggestingthat adjacent spikes were more positively correlated afterthe treatment with picrotoxin (Fig. 6E and F).

Recent studies suggest that subtypes of GABAA receptorsmay differentially contribute to phasic (i.e. synaptic) versustonic inhibitory conductances in the central nervoussystem (Bai et al. 2001; Semyanov et al. 2003; Farrant& Nusser, 2005), including SON neurons (Park et al.2006). Tonic inhibitory conductances can decrease inputresistance, affecting the size and duration of voltage

Figure 4. The GABAergic activity recorded in coronal hypothalamic slices was largely from miniatureIPSCs (mIPSCs), which are more frequent in OT neuronsRecordings were made with a pipette solution containing 120 mM CsCl to maximize Cl− currents. Ten micromolarDNQX and 40 μM AP5 were used to block glutamatergic activities and isolate IPSCs. Aa and b, mIPSCs wererecorded at −60 mV as inward currents; the 2 traces are from the same OT neuron before and after applying0.5 μM TTX. Inset Ac, expanded traces indicate a doublet (right arrow) and a putative monoquantal event (leftarrow). Scale for expanded traces: 100 pA/20 ms. Ba and b, mIPSCs were recorded at −60 mV as inward currents;the 2 traces are from the same VP neuron before and after applying 0.5 μM TTX. C, neither the frequency of allIPSCs (a) nor putatively monoquantal IPSCs (b) was different after applying TTX (grey) (n = 18). D, data as in C,for VP neurons. Total IPSCs (Da) and putative monoquantal IPSCs (Db) were unaffected by TTX. Note that the OTneurons in C have many more mIPSCs than do VP neurons in D. Each box plot is composed of 5 horizontal linesthat represent the 10th, 25th, 50th, 75th and 90th percentiles of the variable.

response to any injected current. High concentrations ofpicrotoxin (100 μm), as well as gabazine (10 μm) andbicuculline (20 μm), can block both phasic and tonicinhibition. In contrast, a low concentration of gabazine(1 μm) exclusively blocks phasic inhibition (Semyanovet al. 2003). We also tested the presence of tonic inhibitionin SON neurons and found this current in OT neurons(Fig. 7). For VP neurons, tonic inhibition was difficult toassess owing to the highly unstable holding baseline whenusing this high-Cl− internal solution, although a previousreport suggests that this current is present in VP neuronsas well (Park et al. 2006).

Since 100 μm picrotoxin would block both GABAergicsynaptic activity and tonic inhibition (as well as other Cl−



channels, such as those associated with glycine receptors),the effect of the more specific blockade of GABAergicsynaptic (phasic) activity was tested with 1 μm gabazine.As with picrotoxin, the CV of ISIs was significantlyreduced after gabazine in OT neurons (Fig. 8C). Althoughthe Fano factor, the other firing variability parameterexamined, was not significantly different after gabazine,the tendency was the same as CV. In addition, theinitial peak of averaged serial correlogram increasedafter gabazine for OT neurons (control, 0.112 ± 0.144;gabazine, 0.261 ± 0.119; P = 0.007). Interestingly, for VPneurons only the initial peak of averaged serial correlogramincreased after gabazine (control, 0.249 ± 0.167; gabazine,0.329 ± 0.143; P = 0.033). Input resistance was slightlybut significantly decreased after gabazine (Fig. 8D). If theblockade of the few IPSCs possessed by VP neurons canaffect input resistance, it is more likely that input resistancewould increase instead of decrease after blockade. Thus,this finding remains anomalous. It does, however, suggestthat changes in input resistance per se are insufficient toaffect variability as measured herein, and also that toniccurrent may have an added role in VP neuron firingvariability, since picrotoxin did reduce both CV and Fanofactor in VP neurons.

Effects of blockade of EPSCs on continuouslyfiring VP and OT neurons

In a separate group of neurons, DNQX (10 μm) wasapplied to selectively block fast EPSCs. In contrastto the effects of blocking inhibitory transmission, wefound no significant differences in firing variability

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CsCl to maximize Cl− currents. Ten micromolar DNQX and 40 μM AP5 were used to block glutamatergic activitiesand isolate IPSCs. B, intersynaptic, all events histogram of IPSCs from the full 1 min recording period, fitted with asingle exponential function. C, serial correlogram, showing no relationship among the intersynaptic intervals over200 orders.

(Table 2) or firing rate (not shown) for either type ofneuron. However, the input resistance was significantlyreduced after DNQX for both OT (1100 ± 271 versus973 ± 302 M�; n = 10; P = 0.022) and VP neurons(741 ± 252 versus 621 ± 276 M�; n = 10; P = 0.022). Itis difficult to explain this reduction in input resistance,since a conductance block should have increased, notdecreased input resistance. Furthermore, the blockade ofIPSCs failed to significantly increase input resistance, andthere were more IPSCs than EPSCs. Since DNQX wasdissolved in 0.1% DMSO, we also tested for vehicle effectson input resistance in 14 neurons, but the results were notsignificant (P = 0.08).

Since DNQX had an effect that appeared unrelatedto its blockade of fast EPSCs, we also tested withkynurenic acid (2 mm), a broad-spectrum antagonist ofAMPA/kainate and NMDA receptors. Kynurenic acidalso blocked virtually all EPSCs, yet had no significanteffect on input resistance in either OT (856 ± 426versus 758 ± 488 M�; n = 8; P = 0.21) or VP neurons(624 ± 234 versus 626 ± 261 M�; n = 8; P > 0.99). Nochanges were found in the CV, Fano factor (Table 2,DNQX) or firing rate (not shown) for either cell type. Theinitial peak of the averaged serial correlogram likewise wasnot altered by kynurenic acid in either OT or VP neurons(not shown). These results confirm that the reductionof the input resistance observed in both cell types withDNQX was unrelated to the blockade of AMPA receptors,or EPSCs, and also that this reduction did not influencefiring variability. Thus, in these coronal slices, blockade ofEPSCs had little effect on the irregular/continuous firingpattern of either cell type, probably because of their relativeinfrequency compared with IPSCs.



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Figure 6. Oxytocin and VP neurons fire more regularly after the application of picrotoxinAa, the control firing of an irregular OT neuron. Ab, the expanded part of the trace indicated in Aa. Ba, firing afterapplying 100 μM picrotoxin. Bb, the expanded part of the trace indicated in Ba. Note the increased bouts of regularISIs and the relative lack of synaptic noise. C, firing variability is decreased in OT neurons following picrotoxin. Boththe Fano factor (P = 0.022) and the CV (P = 0.006) were reduced after the application of picrotoxin. The firingrate (P = 0.363) and input resistance (P = 0.155) did not change significantly (n = 14 for Fano and CV, 11 forRin). D, similar results were found in VP neurons (Fano factor, P = 0.030; CV, P = 0.030; firing rate, P = 0.986;Rin, P = 0.198; n = 21 for Fano and CV, 19 for Rin). E, the averaged serial correlogram of 14 OT neurons showsthat after picrotoxin the initial peak increased (P = 0.03 for adjacent pairs of ISIs), suggesting that the spikes aremore positively correlated over short periods. F, the averaged serial correlogram of 21 VP neurons shows a similarincrease in the initial peak after picrotoxin (P = 0.05). For clarity, the error bars in E and F are S.E.M.



Phasic firing

As shown in Figs 2 and 9, phasic bursting neuronsexhibited long periods of activity separated by comparablesilent periods. We analysed 22 cells that showed phasicfiring in the control state to give a baseline of the propertiesof phasic firing (Table 3) using the criteria listed in theMethods. The data are comparable to a recent in vitrostudy using extracellular recordings in slices (Sabatieret al. 2004), with the exception that intraburst firing ratesreported here are relatively low, perhaps because a lowerextracellular potassium concentration (2.5 versus 6.2 mm)was used in the present study.

The expression of phasic firing within any neuron waslabile over the recording period and thus it was difficultto quantify drug effects; some neurons were phasic only inthe control period, some only during drug treatment andothers in both periods. We applied picrotoxin (100 μm)to eight phasic bursting neurons. Three of these neuronsretained phasic activity, with little difference from thecontrol condition, whereas five exhibited continuousactivity during drug treatment. However, another eightneurons that fired in the irregular/continuous patternduring the control period adopted phasic firing duringpicrotoxin application. A similar difficulty was observedwith glutamate receptor blockade. Five phasic neuronswere tested with DNQX (10 μm) and, of these, fourfired continuously following drug treatment, with onlyone maintaining phasic activity. However, another threecontinuously firing neurons adopted phasic activityfollowing AMPA receptor blockade. Of two phasic burstingneurons tested with kynurenic acid, both maintainedthis discharge pattern in response to drug; another fourneurons firing in the continuous/irregular mode adoptedphasic bursting in response to kynurenic acid. Owing tothese inconsistencies, we tried to discern how synapticactivity in general might affect phasic activity by applying acombination of picrotoxin (100 μm) and DNQX (10 μm)to block IPSCs and EPSCs simultaneously in severalphasic bursting neurons. Of eight phasic neurons tested,five neurons fired phasically both before and after thecombined application of picrotoxin and DNQX, and threephasic neurons fired continuously after this combinedtreatment.

Since picrotoxin but neither DNQX nor kynurenicaffected the firing parameters of irregular/continuousfiring, for statistical purposes we pooled the fivephasic firing neurons from combined synaptic blockers(picrotoxin and DNQX) with an additional three neuronsexposed only to picrotoxin, all eight of which maintainedthe phasic pattern in both periods (Fig. 9). We detectedno significant differences in any of the firing parameters:intraburst firing rate, intraburst CV, intraburst Fano factor,proportion of time active, burst frequency, burst length,silence frequency and silence length (Fig. 9). Thus, in some

neurons at least, phasic patterning was not influencedstrongly by inhibitory synaptic activity.

Could the difference between the responsiveness topicrotoxin of continuously firing and phasic VP neuronsbe related to different amounts of inhibitory synaptic inputduring the control period? Indeed, the 21 VP neurons thatfired continuously throughout the experiment, and wereexposed to picrotoxin, had almost three times as manyIPSCs (mean (x) = 2.4 ± 1.98 Hz) as the eight phasicneurons tested with picrotoxin or the cocktail of DNQXand picrotoxin (mean (x) = 0.92 ± 1.05 Hz; P = 0.034),but did not differ in EPSC frequency (P = 0.86). Theseresults indicated that in the control state, the firing patternadopted by VP neurons might relate to the frequencyof spontaneous inhibitory inputs. We tested this further

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CsCl to maximize Cl− currents, at a holding voltage of −60 mV. Fortymicromolar AP5 and 10 μM DNQX were added to the ACSF to inhibitionotropic glutamate receptors, and 0.5 μM TTX was added to blockvoltage-sensitive Na+ channels. A, an OT neuron was first exposed to1 μM gabazine for 3 min to block mIPSCs. After some recovery ofmIPSCs during the wash, 100 μM picrotoxin was applied for 3 min.Not only were mIPSCs blocked, but also the holding current wasoutwardly shifted. The dashed line indicates the holding current in theabsence of picrotoxin. B, box plot of the holding current from 19 OTneurons at 4 periods. Differences were detected between controlholding current after wash and after the application of picrotoxin.



by comparing the frequency of IPSCs in the controlstate of VP continuously firing neurons (n = 21) andall phasic bursting neurons (n = 22). Although the VPcontinuously firing neurons tended to have more IPSCs(mean frequency of IPSCs, 2.40 ± 1.98 Hz) than thoseneurons with phasic firing in control states (mean

Figure 8. Gabazine regularized the firing patterns of OT neurons in virgin ratsAa, instantaneous frequency during control state. Each dot represents the reciprocal of the corresponding interspikeinterval. Notice the wide dispersion of the dots. Ab, 25 s sample from the spike train represented. Notice thevariability of ISIs. Ba, instantaneous frequency graph after 1 μM gabazine. Compared with Aa, the dots are morecondensed, representing a reduction of variability of firing after gabazine. Bb, 25 s sample from the spike train.Notice the regularity of ISIs. C, for OT neurons, CV of ISIs was significantly reduced after gabazine. Firing rate andinput resistance were not affected by gabazine. D, for VP neurons, the firing variability (Fano factor and CV) andfiring rate were not affected by gabazine, but the input resistance was slightly decreased.

frequency of IPSCs, 1.55 ± 1.72 Hz), the difference was notsignificant (P = 0.09).

We then checked whether the expression of phasicactivity was related to drug treatment on a broader scale.A contingency table analysis of the irregular/continuousversus phasic patterns before and after drug



Table 2. Lack of effect of DNQX (10 μM) or kynurenic acid (KA; 2 mM on firing variability of continuously firing OT and VPneurons

Drug Type (n) CV Control CV Drug (P) Fano Control Fano Drug (P)

DNQX OT (10) 0.82 ± 0.50 0.81 ± 0.38 (0.24) 0.59 ± 0.38 0.58 ± 0.28 (0.51)VP (10) 0.23 ± 0.05 0.26 ± 0.11 (0.24) 0.13 ± 0.04 0.17 ± 0.11 (0.11)

KA OT (9) 0.46 ± 0.32 0.38 ± 0.14 (0.59) 0.29 ± 0.25 0.23 ± 0.10 (0.59)VP (10) 0.17 ± 0.03 0.20 ± 0.06 (0.28) 0.09 ± 0.02 0.12 ± 0.05 (0.07)

Values are means ± S.D.

treatment revealed no tendency for VP neurons to firephasically in control versus synaptic blockade (contingencytable correlation test: P = 0.217). Thus, synaptic activity,or its relative absence, was not strongly related to theexpression of phasic bursting.

Figure 9. Phasic firing was not affected by synaptic blockadeA1, phasic bursting SON neuron in control ACSF for a 5 min recording. Ab, ratemeter of spike activity in Aa. Thered lines in Ab and Bb indicate bursts detected by the computer program. The first firing period was not analysedbecause the initiation of burst required at least 3 s of preceding silence (see Methods). The blue line indicates thesilent period detected by the program. The silent period following the last burst was not counted because theduration of the silent period is unknown. Notice that isolated spikes (the arrows in Aa and Ba) were consideredpart of the silent period between adjacent bursts. Ba, trace from the same neuron as in A after the applicationof picrotoxin and DNQX. Bb, ratemeter from neuron shown in Ba. See A for explanation. C, D and E show thestatistical data from 8 neurons that exhibited phasic activity before and after synaptic blockade (5 were treatedwith combined picrotoxin and DNQX; 3 were treated with picrotoxin only). There were no significant differencesbetween control and synaptic blockade periods for intraburst CV (C), intraburst firing rate (D) and the proportionof active time (E). We also compared the length of bursts and interburst intervals between the 2 periods and foundno significant differences. Burst length of control, 33.5 s; burst length of drug treatment, 42.2 s; silence length ofcontrol, 25.4 s; and silence length of drug treatment, 32.3 s.

Differences between OT and VP neurons

Although the CVs of both OT and VP irregularly/continuously firing neurons were responsive to picrotoxin,we noted differences in the control firing patterns



Table 3. Parameters of the phasic firing pattern (n = 22)

Parameter Value (mean ± S.D.)

Mean burst duration 39.9 ± 7 sMedian burst duration 33.3 sBurst duration range 12–167 s

Burst frequency 3.6 ± 0.4 per 5 minMean silence duration 31.4 ± 4.4 sMedian silent duration 29.1 sSilence duration range 7.5–83 s

Silence frequency 4 ± 0.4 per 5 minMean intraburst firing rate 3.4 ± 0.3 Hz

Mean intraburst CV 0.435 ± 0.017Proportion of time active 37.6 ± 3.5%

of these two cell types (Fig. 10). The two firingvariability parameters, CV (P < 0.0001) and Fano factor(P < 0.0001), were significantly different between OT(n = 33) and VP neurons (n = 41) in the control state,with OT neurons the more variable of the two. In

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Figure 10. Comparison of OT and VP neuronal propertiesA, OT neurons have a larger firing variability, as indicated by the larger Fano factor (P < 0.0001) and CV(P < 0.0001), compared with VP neurons. B, OT neurons fire more slowly than VP neurons near threshold(P < 0.0001). C, averaged serial correlograms showed that the initial peak of VP neurons was larger than thatof OT neurons (P = 0.002), suggesting that for short periods (10 spikes), neighbouring spikes of VP neurons aremore positively correlated than those of OT neurons. D, OT neurons have larger input resistance than VP neurons(P = 0.002). E, OT neurons exhibited more IPSCs (P < 0.0001) and EPSCs (P = 0.042) than did VP neurons. For A–E,n = 41 VP neurons and 33 OT neurons. F, since OT neurons have more IPSCs, we predicted that their response topicrotoxin would be larger than that of VP neurons. Although the tendency was that the variability of firing (Fanofactor and CV) was reduced in OT compared with VP neurons in response to picrotoxin, neither effect reachedstatistical significance (CV, P = 0.07; Fano factor, P = 0.09).

contrast, the minimum mean firing rate of VP neuronsnear threshold was significantly larger than that of OTneurons (P < 0.0001). The input resistance was larger inOT neurons (Fig. 10D; P = 0.002). Adjacent spikes in VPneurons were more positively correlated than those of OTneurons, as detected by the higher initial peak of averagedserial correlation (P = 0.002; Fig. 10C). This effect appearsto persist for 5–10 spikes.

Spontaneous synaptic activity also differed betweenthe two cell types (Fig. 10E). Oxytocin neurons exhibitedmore IPSCs (P < 0.0001) and EPSCs (P = 0.04). Since OTneurons were found to exhibit more IPSCs than did VPneurons and had CVs indicating more firing irregularity,we considered that the effect of picrotoxin on firingpatterns of these two cell types might differ (Fig. 10F).Although suggestive for the two variability indices, thepercentage change following picrotoxin OT (n = 14) andVP neurons (n = 21) on the three firing parameters (CVafter PX minus CV before PX, P = 0.074; Fano after PXminus Fano before PX, P = 0.092; FR after PX minus FR



before PX, P = 0.5) did not differ. The mean change in CVexerted by picrotoxin was −0.175 ± 0.188 for OT neuronsand −0.069 ± 0.135 for VP neurons. The mean change inthe Fano factor for OT and VP neurons was −0.14 ± 0.23and −0.044 ± 0.091, respectively. In addition, there wasno significant difference in the percentage change or inthe actual value of the difference in the serial correlationcoefficient of adjacent spike pairs induced by picrotoxin inthe two cell types (data not shown).

As mentioned previously (Fig. 4), OT neurons alsoexhibited more IPSCs and mIPSCs than did VP neuronswhen using symmetrical Cl− concentrations to enhanceCl−-mediated events. A subset of these mIPSCs was furtheranalysed for differences in amplitude and decay kinetics.For this analysis, putatively monoquantal mIPSCs wereaveraged and fitted with one or two exponentials. Inmost cases, the fit required two exponentials, and weweighted the two for an averaged time constant (τ ) basedon the relative amplitude coefficients. No differences werefound in the averaged mIPSC decay (OT, 9.9 ± 2 ms; VP,8.9 ± 1.6 ms; P = 0.229) or in the averaged peak mIPSC(OT, −130 ± 5 pA; VP, −138.8 + 48.9 pA; P = 0.578)between the two cell types.

Discussion

In the present study, we demonstrated that blockadeof Cl−-mediated inhibitory currents in the coronalhypothalamic slice shifts firing variability along theirregularly/continuously firing axis of OT and most VPneurons when the membrane potential is near spikethreshold. This change was independent of any consistentchange in firing rate or input resistance, and was notevident when blocking EPSCs with DNQX or withkynurenic acid. Thus, blockade of Cl− channels mediatingboth tonic and synaptic currents with picrotoxinredistributed spikes over long periods of time, withoutchanging their number. This conclusion was furtherconfirmed in OT neurons using 1 μm gabazine, whichselectively blocked GABAergic synaptic activity. Thislatter result suggests that one consequence of randomlydistributed mIPSCs is to distribute spikes to more irregularpatterns, which previous studies revealed can lead to shortperiods of enhanced hormone release when spikes areclustered (Cazalis et al. 1985; Bicknell, 1988).

GABAergic transmission in SON

While a minority of neurons probably get local input fromthe perinuclear zone in this preparation (Wuarin, 1997),previous studies have shown little effect of TTX on synapticactivity, either excitatory or inhibitory, indicating a paucityof spontaneously active SON afferents in the coronal slice(e.g. Kabashima et al. 1997; Brussaard et al. 1999). Inagreement, we found that the frequency and amplitude of

IPSCs were unaffected by TTX in both OT and VP neurons,and that the distribution of IPSCs was largely random. Inaddition, we found for the first time that: (1) spontaneousIPSCs predominated over EPSCs in vitro; and (2) OTneurons exhibited far greater numbers of IPSCs whencompared with VP neurons. Morphologically, GABAergicinnervation of OT and VP neurons appears roughly similar(Theodosis et al. 1986). Thus, if GABAergic synaptic inputis comparable, our results suggest that the probabilityof spontaneous GABA release is much smaller onto VPthan OT neurons, at least in virgin female rats. Whetherthis is related to intraterminal mechanisms governing thisactivity or some differential, tonic activation of presynapticreceptor is presently under investigation.

Previous studies have shown that the precise role ofGABAergic activity in controlling OT neurons, at leastduring lactation, is complex. The GABAA receptor agonistmuscimol and the antagonist bicuculline can inhibitthe milk ejection reflex after intracerebroventricularapplication in vivo, and the background firing of bothOT and VP neurons was found to be inhibited byboth compounds (Voisin et al. 1995). In contrast, intra-venous injection of bicuculline methochloride increasedOT release during late pregnancy, and increased firingrate when applied to brain slices from late pregnant rats(Brussaard et al. 1997). Unfortunately, in the in vivoexperiments, there is no way to determine the site of actionof these drugs. Furthermore, both studies used bicucullinesalts, which block the small conductance K+ channel(SK)-mediated, Ca2+-dependent after-hyperpolarizationin central neurons (Debarbieux et al. 1998); these channelsare prominent on MNCs and strongly gate firing rate(Renaud & Bourque, 1991).

Certainly, the degree of spontaneous synaptic activitywould be expected to be larger in vivo (Bourque & Renaud,1991). In slices, connections are obviously removed anddendrites truncated. The localized application of GABAA

antagonists (picrotoxin or gabazine) near recorded SONneurons moderately increased the background firing rateyet interrupted the milk ejection reflex, whereas theapplication of GABA and the GABA agonist isoguvacinedecreased the basal electrical activity but facilitated thereflex (Moos, 1995). During lactation, irregular firingprecedes burst firing during the milk ejection reflex, andthe ability of GABA to facilitate milk ejection bursts iscorrelated with its promotion of irregular firing (Brownet al. 2000). Since we have found that spontaneousIPSCs impart irregularity to the spike train near spikethreshold, we suggest that one mechanism for the switchto irregular firing during background firing periodscould be an increase in GABAergic activity. While thespecific sources to OT or VP neurons are incompletelydescribed, GABAergic inputs to the SON are thought toderive from multiple forebrain and hypothalamic sources(Roland & Sawchenko, 1993). The perinuclear zone of the



SON possesses a substantial complement of GABA- andglutamic acid decarboxylase (GAD)-positive cell bodiesthat could support a local inhibitory projection system(Theodosis et al. 1986; Jhamandas et al. 1989; Roland &Sawchenko, 1993; Wuarin, 1997). This zone of GABAergicneurons is thought to mediate the inhibition of VP neuronsfollowing transient hypertension, possibly via a projectionfrom the diagonal band of Broca (Jhamandas et al. 1989).

It has been proposed that GABAA-receptor signallingcan powerfully influence the firing of OT neurons ina reproductive-state-specific manner (Brussaard et al.1999). These changes include transient increases in mIPSCfrequency during early lactation that might reflect synapticplasticity (El Majdoubi et al. 1997), and pregnancy-relatedchanges in the neurosteroid modulation of postsynapticGABAA-mediated synaptic transients, each of which coulddynamically alter inhibition to influence OT release. Ifthe present results have bearing on this plasticity, wemight expect that GABAergic synaptic activity could exerteven greater influence over spike train variability duringdifferent reproductive states.

In suprachiasmatic nucleus (SCN) neurons, blockade ofIPSPs by bicuculline or gabazine also converted irregularfiring to a more regular firing pattern, but in the case ofbicuculline, also depolarized the neurons and increasedfiring rate (Kononenko & Dudek, 2004). However,the depolarization and perhaps the strong increase infiring rate were probably due to the non-specific effectsof bicuculline salts, known to block Ca2+-dependentafter-hyperpolarizations, or even to their ability to inhibittonic currents. Like SCN neurons, the regularization ofspike trains in OT neurons was still found after selectiveIPSC blockade with gabazine.

Glutamatergic transmission in the SON

Although we found that EPSCs contributed little to spikedistribution, this is most likely to result from their lowfrequency in this preparation and should not be inter-preted as evidence for a diminished role for glutamatein producing patterns of activity in either cell type.Indeed, glutamate clearly plays an important role inthe generation of OT release during lactation (Parker &Crowley, 1993) and, in certain in vitro models, burstingactivity similar to that observed in vivo either can beinduced pharmacologically in hypothalamic slices withnoradrenaline (Wang & Hatton, 2004, 2005) or is presentspontaneously in organotypic cultures (Jourdain et al.1996; Israel et al. 2003) and, in both cases, is dependenton glutamatergic transmission. Likewise, phasic activity inVP neurons in vivo is suppressed or even blocked in thepresence of glutamate receptor antagonists such asketamine (Nissen et al. 1994) and CNQX (Nissen et al.1995; Brown et al. 2004), even in the face of osmoticsimulation.

Could the difference between IPSC and EPSC frequencyhave a morphological basis? GABAergic synapses arethought to account for 45–50% of all terminals in theSON (Decavel & Van den Pol, 1990), almost twice that ofglutamatergic synapses (El Majdoubi et al. 1997). However,even this difference is less than the striking differencewe found between IPSC and EPSC frequency. Perhapsfactors controlling spontaneous release differ between thetwo terminal types, factors that relate to terminal releasemachinery or to tonic activity from a host of neuroactivesubstances known to act presynaptically in the SON, suchas glutamate, GABA, OT and VP, among others.

Firing patterns in vitro

With the exception of milk ejection bursts, the firingpatterns of MNCs evident in vivo (Poulain et al. 1988) areroutinely present in vitro. In vivo, the slow irregular firingpattern characterizes both cell types when not activated,whereas continuous firing is more associated with OTneurons and phasic bursting with VP neurons (Poulainet al. 1988). However, continuously firing VP neuronsare not uncommon in vivo (Renaud et al. 1987), andindividual VP neurons can even exhibit this and the phasicpattern during the same recording period, depending onstimulation level (Poulain & Wakerley, 1982). Althoughwe found that a majority of VP neurons exhibit phasicbursting activity at some point during long recordings,many exhibited only slow irregular/continuous activity.Thus, as we previously found in male rats (Armstrong et al.1994), continuous activity is not a signature of OT neuronsin vitro. Nevertheless, we found distinct differences inthis pattern near threshold in the two cell types, inthat OT neurons were slower and more irregular than(non-phasic) VP neurons. This irregularity was indicatednot only by the higher CV of OT neurons, but also bytheir lower initial peak of the averaged serial correlationcoefficients compared with VP neurons. We suggest thatboth of these factors are related to the increased amount ofinhibitory input impinging on OT neurons. In addition,the fewer IPSCs on VP neurons, as judged by the responseto gabazine, do not contribute as much to variabilityalong the irregularly/continuously firing continuum (onlythe serial correlation coefficient was altered). This leadsus to conclude that picrotoxin, which more consistentlyaffects variability in VP neurons, may do so throughthe additional blockade of a tonic current. However,since input resistance was not significantly increased bypicrotoxin in these neurons, these effects are either subtleor perhaps occur on dendrites, electronically remote fromthe soma. Interestingly, although each cell starts on averagefrom a different CV, firing rate and tonic GABAergic input,picrotoxin has about the same magnitude of effect on CVin the two cell types. Although in vivo VP neuronal spiketrains are reportedly more variable than those from OT



neurons (Bhumbra & Dyball, 2004), this is explained bythe authors by their inclusion of the interburst intervalsof phasic activity of VP neurons for the calculation of CV,which weights long intervals at the expense of shorter ones.

Frequency and variability in the background spike trainhas physiological meaning for OT neurons during milkejection. Injection of OT into the third ventricle increasedbackground discharge of OT neurons and stronglyfacilitated the milk ejection reflex (Freund-Mercier &Richard, 1984). However, systemic hypertonic saline(Negoro et al. 1987) produced a strong and sustainedincrease in background activity, which delayed theoccurrence of the following burst. Brown & Moos (1997)found that an intermediate range of background firing,from 1 to 3 Hz, was optimal for promoting the milkejection burst. Subsequent analyses revealed that thevariability of the background firing was a critical factor inpredicting bursts (Brown et al. 2000; Moos et al. 2004).Within 1–2 min prior to a milk ejection burst, back-ground firing rate increased and became irregular. Furtherincreasing the firing rate, e.g. with hypertonic saline,reduced variability and inhibited milk ejection bursts,much as Negoro et al. (1987) found. Our data suggest thatinhibitory activity, perhaps both tonic and synaptic, canalter variability and could be a factor that governs bursting.Indeed, GABA itself can reduce firing rate but increase theprobability of bursting. This may be even more likely withexpression of the sustained outward rectifier and rebounddepolarization of OT neurons, which are sensitive to smallhyperpolarizations from near spike threshold (Stern &Armstrong, 1995).

Phasic bursting neurons, however, were inconsistentlyaffected by GABAergic and glutamatergic synapticblockade. Consistent with previous studies in vitro(Andrew & Dudek, 1983; Hatton, 1982), this suggeststhat phasic firing per se in VP neurons does not requiresynaptic input in vitro. However, we caution over-interpretation of this result; our neurons were often heldnear spike threshold with current injection, allowing theactivation of voltage-dependent conductances, such asthe spike depolarizing after-potential (Andrew & Dudek,1983; Bourque, 1986), that are critical to the expressionof phasic activity. Since most VP neurons are slowfiring and non-phasic in vivo under resting conditions(Poulain & Wakerley, 1982), activation through membranedepolarization is achieved synaptically or through directosmotic depolarization. Indeed, recent evidence suggests aprofound dependence of phasic activity on glutamatergicsynaptic transmission in vivo (Nissen et al. 1995), even inthe face of strong osmotic stimulation (Brown et al. 2004).Thus, osmotic depolarization achieved from the intrinsicosmoreceptivity of VP neurons in vivo is insufficientto sustain phasic activity in the absence of additionalafferent excitatory drive. The present results that showlittle influence of synaptic activity on phasic activity also

should be viewed in the context of two additional caveats:firstly, that phasic activity is labile over a recording periodregardless of the presence of spontaneous synaptic activityand, secondly, that intracellular current injection is a steadyforce, unlikely to be replicated by neuronal factors invivo. Our results do suggest, however, that ISIs duringphasic bursts in vitro are temporally dispersed, largelyfrom intrinsic rather than synaptic factors and, further,that these properties lead to a more regular firing patternwithin bursts. This is consistent with the results of Sabatieret al. (2004), who found that trains within bursts in vivohave a more random distribution of spike times comparedwith the more regular firing neurons recorded in vitro.

Phasic (synaptic) inhibition and tonic inhibition

We confirmed the presence of a phasic (mediated byGABAergic synaptic transmission) and tonic inhibition inOT neurons. Tonic inhibition has been reported to affectfiring properties in other neurons. In cortical interneurons,which express tonic GABAergic currents, the applicationof 100 μm picrotoxin increased the firing rate producedby injecting any particular current in current-clampmode, probably reflecting an increased input resistance(Semyanov et al. 2003). A similar result was reported incerebellar granule cells using a higher dose of gabazine(10 μm), which blocked both IPSCs and GABAergic toniccurrents (Brickley et al. 2001). In the meantime, theamplitude and duration of sEPSPs in cerebellar granulecells were enhanced by 10 μm gabazine, which suggestedthat tonic activation of GABAergic receptors altered theresponse of granule cells to excitatory inputs (Brickley et al.2001). In vivo whole-cell patch clamp recording showedthat topically applied gabazine (0.5 mm) also produceda significant leftward shift in the spike frequency versusinjected current curve in cerebellar granule cells, consistentwith the in vitro studies (Chadderton et al. 2004).

According to Park et al. (2006), the blockade ofGABAergic tonic currents induced similar effects in MNCsto those in other neurons previously tested, i.e. increasedinput resistance leading to a shift in the current–frequencycurve. These tonic currents were not mediated by glycinereceptors. For silent SON neurons, the application ofpicrotoxin or coapplication of gabazine and bicuculline,but not gabazine alone, depolarized the neuron to increaseaction potential firing (Park et al. 2006). Tonic inhibitionwas observed in both OT and VP neurons, whereas inthe present study, tonic inhibition was not assessed inVP neurons owing to the highly unstable holding base-line. In addition, we did not find a consistent increase ininput resistance to picrotoxin, though the tendency was inthat direction, but we did find effects of picrotoxin in VPneurons that were not repeated with gabazine. Together,these studies suggest that tonic inhibition is present inMNCs in addition to phasic inhibition, and that it can



modulate neuronal electrical properties in MNCs, as inother neurons. We avoided the use of bicuculline in thisstudy because its salts are known to block Ca2+-dependentK+ currents mediated by SK channels, as are found inthe SON and which mediate spike after-hyperpolarizations(Debarbieux et al. 1998).

Conclusion

In summary, we demonstrated that inhibitory activitiescould affect the firing patterns of both VP and OT neuronsin vitro, and that OT neurons were different from VPneurons in several respects, but especially in that theyexpressed a greater spontaneous GABAergic synaptic inputthat in turn contributed a more consistent influenceon firing variability. To achieve better insight into thedifferential contribution of phasic (synaptic) inhibitionand tonic inhibition to the firing patterns of these twocell types, the restoration of phasic inhibitory conductanceand/or tonic inhibitory conductance with dynamic clamp(Mitchell & Silver, 2003) would be useful. Finally, it willbe important to determine to what degree the synapticplasticity associated with reproductive state influencesfiring properties.

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Acknowledgements

We thank Drs Joseph C. Callaway, Ryoichi Teruyama and Talent

Schevchenko for their help in this study, and Dr Peter Roper for

his comments on a previous version of the manuscript. This work

was supported by NS23941 (NINDS) and HD41002 (NICH/HD)

to W.E.A.

Supplemental material

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