all experimental procedures conform to the uk animals ( scientific procedures ) act 1986 and to ethical standards set out by the university of leeds ethical review committee . 1012 day old wistar rats ( n=89 ) of either sex were deeply anaesthetized with urethane ( 2 g / kg of body weight intraperitoneally ) . animals were transcardially perfused with ice cold sucrose artificial cerebrospinal fluid ( sucrose acsf ) containing ( in mm ) : sucrose ( 215 ) , nahco3 ( 13 ) , glucose ( 5 ) , kcl ( 1.5 ) , nah2po4 ( 1.5 ) , mgso4.7h2o ( 1 ) and cacl2 ( 1 ) , then humanely killed by decapitation . the thoracic spinal cord was dissected out and 500 m transverse slices cut using a vibraslice ( campden instruments ) . slices were stored at room temperature submerged in a holding chamber of acsf containing ( in mm ) : nacl ( 124 ) , nahco3 ( 26 ) , glucose ( 10 ) , kcl ( 3 ) , nah2po4 ( 2.5 ) , mgso4.7h2o ( 2 ) , cacl2 ( 2 ) , equilibrated with 5 % co2/ 95% o2 . slices were allowed to equilibrate for at least 20 min in a custom built  interface  recording chamber , superfused with gassed acsf at 3035 c . to keep slices moist , humidified 95 % o2/ 5% co2 was circulated in the recording chamber . a glass micropipette ( 24 m tip diameter ) signals were amplified 10 by an axoclamp2b amplifier ( axon instruments , usa ) , and a further 1000 amplification was achieved with two neurolog nl106 ( digitimer , uk ) amplifier modules . signals were low - pass filtered ( < 35 hz ) , notch filtered for 50 hz mains noise ( humbug , digitimer ) , digitized ( ced1401plus , cambridge electronic design , uk ) and captured at 5 khz on a pc running spike2 software ( cambridge electronic design , uk ) . extracellular field potential recordings were made , at depths of approximately 100 m below the surface , from the iml of 157 transverse spinal cord slices ( which retain the mediolateral dendrites that are predominant in spns at this age ( markham and vaughn , 1991 ) ) from 89 neonatal rats . extracellular iml activity had peak amplitudes of 2060 v which did not vary appreciably during the course of a recording . background noise levels were determined by comparing traces captured with the electrode placed in the iml ( fig . the amplitude of the activity was much lower with the electrode in the bath ( < 10 v ) , as expected . 1a , b ) revealed that the frequency components of the two traces were different : the background noise consisted mainly of a small peak at 3 hz , whereas a larger proportion of higher frequencies was present in the neuronal trace . to enable post hoc verification that the recording electrode was correctly positioned , power spectra ( fast fourier transform ( fft ) with hanning window , fft size=8192 and resolution=0.6 hz ) and autocorrelograms were generated from 1 min of activity . the field potentials were considered to be rhythmic if there was a peak in the power spectrum and the first autocorrelogram peak to the right of the central peak indicated a correlation coefficient of greater than 0.1 . the principal frequency of rhythmic activity was assessed from the highest peak in the power spectrum . the degree of rhythmicity (  area power  ) was quantified by measuring the area under the power spectrum in a 3 hz band surrounding the highest peak . note that due to this analysis protocol , slices that lacked rhythmic discharges nevertheless had an area power considerably greater than zero because a degree of activity in the frequency band of interest is to be expected at all times . if the frequency of the highest peak changed following drug treatment , the power under this new peak was calculated , not the power at the original frequency . if no power spectrum peak was present ( for example , after blockade of oscillations with a drug ) , then area power was measured from the same 3 hz range as in control conditions . statistical comparisons involving area power were carried out with wilcoxon matched pairs signed rank test because these data do not fit a normal distribution . frequency data are presented as meanstandard error of the mean ( sem ) , whereas area power data are presented as median and interquartile ( iq ) range . stock solutions of drugs ( apart from 5-ht which was made up fresh on the day ) were diluted in acsf on the day of the experiment and superfused over the slice without recirculation . drugs were obtained from tocris bioscience ( uk ) except where stated : 5-hydroxytryptamine hydrochloride ( 5-ht , sigma - aldrich , uk ) , methyl-5-hydroxytryptamine maleate ( me5-ht ) , 6-chloro-2-(1-piperazinyl)pyrazine hydrochloride ( mk212 ) , potassium ( 2s,4as,6ar,6as,6br,8ar,10s,12as,14br)-10-hydroxy-2,4a,6a,6b,9,9,12a-7 heptamethyl-13-oxo-3,4,5,6,6a,7,8,8a,10,11,12,14b - dodecahydro-1h - picene-2-carboxylate ( 18-glycyrrhetinic acid , stock in 50% ethanol ; sigma - aldrich , uk ) , ( as)-rel - a-(2r)-2-peperidinyl-2,8-bis(trifluoromethyl)-4-quinolinemethanol monohydrochloride ( mefloquine ) , n-[2-[[3-(dimethylamino)propyl]thio]phenyl]-3-phenyl-2-propenamide hydrochloride ( cinanserin ) , tetrodotoxin citrate ( ttx ) , bicuculline hydrochloride ( bicuculline ) . power spectral and autocorrelation analysis of iml activity revealed spontaneous rhythmic field potential oscillations in 48/164 ( 29.3% ) slices tested . the rhythmic discharges were manifest as a peak in the power spectrum in the range 7.522 hz ( mean 13.50.7 hz ) and as a sinusoidal autocorrelogram ( e.g. fig . oscillation frequency did not drift appreciably during the recording , even though different slices showed a range of frequencies . this rhythmic activity typically persisted for the duration of a recording , which could be in excess of 3 h. of the slices that did not exhibit spontaneous rhythmic activity , a further 18 previously non - rhythmic slices could be pharmacologically manipulated with 5-ht agonists to induce this activity ( see below ) . the other slices were not able to generate spontaneous or 5-ht - induced oscillations ( see discussion ) and therefore were not considered any further . in six slices where robust rhythmic oscillatory activity was recorded from the iml , moving the electrode to regions outside the iml resulted in a loss of rhythmic activity in all slices tested . these regions included the motor nuclei ( lamina ix ) of the ventral horn ( n=3 ) or superficial dorsal horn ( n=3 ) , ( fig . 1c ) and suggests that this particular rhythm was specific to the iml ( see discussion ) . the positioning of the electrode in the iml was verified by deposition of rhodamine from the electrode at the end of recording ( fig . this was seen as the disappearance of the peak in the power spectrum and the flattening of the sinusoidal morphology of the autocorrelogram ( fig . , ttx significantly reduced the power of iml oscillations , from a median value of 45.8 ( iq range 31.667.1 v ) to 4.6 ( iq range 1.76.2 ; p=0.028 , n=6 ; fig . the frequency of the oscillations ( measured at the latest time the power spectrum peak was still detectable ) was unaffected by ttx ( 14.31.5 hz in control conditions vs. 12.31.4 hz in ttx , p=0.201 , n=6 ; fig . sympathetic preganglionic neurons in the iml express the gap junction channel subunit connexin 36 ( cx36 , marina et al . , 2008 ) and fire synchronous action potentials in slices as a result of electrotonic coupling ( logan et al . , we therefore sought to determine whether direct electrical coupling contributed to the rhythmic population activity in our slice preparation . the gap junction blocker 18-glycyrrhetinic acid ( 100 m , verified as effective at this concentration at blocking spikelets , the hallmark of gap junctions in recordings from single spns ; data not shown ) was continuously bath applied to six spontaneously oscillating slices ( fig . 18-glycyrrhetinic acid reduced the area power of spontaneous oscillations by an average of 68.2% ( median power was 42.9 [ iq range 26.956.1 ] v in control and 5.0 [ iq range 4.810.6 ] v in 18-glycyrrhetinic acid , p=0.028 , n=6 ; fig . in addition , the oscillation frequency ( measured immediately before abolition of the power spectrum peak , where necessary ) was significantly reduced by 18-glycyrrhetinic acid from 14.11.0 hz in control conditions to 11.21.5 hz in the presence of the drug ( p=0.02 , n=6 ; fig . another gap junction blocker , carbenoxolone ( 100 m , a concentration at which spontaneous spikelets were reduced in current clamped spns , not shown ) , had a similar effect ( data not shown ) , reducing the power of iml oscillations by an average of 69.8% . area power was reduced from 5.5 ( iq range 3.6110.5 ) v to 1.4 ( iq range 1.036.6 ) v ( p=0.068 , n=4 ) by this drug ; however , this reduction did not quite reach statistical significance , possibly due to the range of ongoing area powers observed . oscillation frequency , measured just before abolition of the power spectrum peak , was not affected ( 15.22.3 hz in control to 16.74.1 hz in carbenoxolone , p=0.47 ) . finally , we also tested the effects of mefloquine , a gap junction blocker which has been reported to block cx36-containing gap junction channels more effectively than those with alternative subunit compositions ( cruikshank et al . , 2004 ) . bath application of 1 m mefloquine , a concentration also verified to reduce spikelet activity in spns ( not shown ) abolished spontaneous oscillations in 2/3 slices ( fig . j ) , and reduced the power from 33.2 to 5.8 v in the remaining slice . the mean reduction in area power produced by mefloquine was 76.4% ( median 33.2 [ iq range 20.240.0 ] under control conditions , 3.9 [ iq range 3.64.9 ] in mefloquine , p=0.109 , n=3 ; fig . oscillation frequency ( measured just before abolition of the power spectrum peak , where necessary ) was not significantly changed by mefloquine , although a decrease from 16.73.1 hz to 12.51.7 hz ( p=0.153 , n=3 ; fig . 3i ) was observed following application of this drug . to test whether inhibitory gabaergic synaptic transmission is required for rhythm generation in the iml , 10 m bicuculline significantly reduced the power of ongoing oscillations from a median value of 19.1 ( iq range 5.135.1 ) v to 3.6 ( iq range 0.84.4 ) v ( average 65.1% reduction  fig . the frequency of the oscillation was not significantly changed by bicuculline ( 11.92.0 hz in bicuculline vs. 10.11.2 hz under control conditions , p=0.149 , n=5 ) . 10m 5-ht was applied to eight slices which were demonstrated to be capable of oscillating , of which five were spontaneously rhythmically active and three were non - rhythmic at the time 5-ht was applied . intermittent application of 5-ht over 30 min using a 5-min - on , 5-min - off protocol resulted in an increase in the area power at the rhythm frequency in all eight slices ( fig . 5 ) . intermittent , rather than continuous , application of 5-ht was employed because this type of protocol was more successful at enhancing inspiratory nerve burst amplitude than continuous application of 5-ht to a brainstem - spinal cord preparation ( lovett - barr et al . , 2006 ) . the increase in area power was from a median value of 4.1 ( iq range 2.110.4 ) v to 14.2 ( iq range 8.734.4 ) v ( average 544.7% increase  fig . the 5-ht - induced rhythmic activity lasted at least 10 min and often in excess of 1 h. 5-ht - induced oscillations fell within the normal range for spontaneous oscillations , averaging 9.91.8 hz . in the five slices which exhibited spontaneous oscillations , the oscillation frequency was not changed by 5-ht treatment ( 9.71.1 hz vs. 9.71.0 hz , p=0.655 , fig . selective agonists were also used to determine the receptor subtype which contributed to the effects of 5-ht . 10 m me5-ht , an agonist of 5-ht2 receptors , mimicked the effects of 5-ht . when applied to slices which were demonstrated to be capable of rhythmic activity , methyl5-ht increased area power in all 12 slices , from a median value of 4.9 ( iq range 2.112.9 ) v to 13.3 ( iq range 5.421.6 ) v ( average 574.0% increase ; fig . 6 ; p=0.005 ) . six of these slices were spontaneously active when the drug was applied , whereas the other six were not . similar to the effects of 5-ht , me5-ht did not significantly change spontaneous oscillation frequency ( 11.71.9 hz during control , 11.31.4 hz during methyl5-ht application , p=0.595 ; fig . in addition , the mean frequency of rhythmic activity induced by me5-ht was very similar to that of spontaneous oscillations ( 13.02.2 hz , p=0.617 ) . since me5-ht does not discriminate between the three subtypes of 5-ht2 receptor , and mrna for 5-ht2c is high in the iml ( fonseca et al . , 2001 ) , the action of the selective 5-ht2c agonist mk212 was tested . 10 m mk212 was continuously bath applied to 12 slices deemed capable of generating rhythmic activity , of which four were spontaneously rhythmic . the area power of iml oscillations was significantly enhanced in all 12 slices ( fig . the median control power in these experiments was 12.0 ( iq range 6.016.0 ) v , rising to 18.7 ( iq range 10.750.2 ) v ( average increase of 167.0% , p=0.006 , n=12 ; fig . 7e ) measured after at least 8 min in mk212 . again , the frequency of spontaneous rhythmic discharges was unaffected ( 15.01.9 hz in control , 16.12.5 hz in mk212 , p=0.288 , n=4 ; fig . 7d ) . to determine the contribution , if any , of 5-ht receptors to the spontaneous oscillations , the 5-ht2 receptor antagonist cinanserin was continuously bath applied to three spontaneously oscillating slices . 10 m cinanserin reduced iml oscillations in all three slices , with a mean reduction in power of 33.3% ( from 0.3550.079 to 0.2360.060 , n=3 ; fig . cinanserin did not significantly affect oscillation frequency : the mean frequency in control conditions was 11.11.2 hz , and in cinanserin the frequency was 9.81.8 hz , n=3 . in this study we have recorded field potentials , through electrodes placed in the iml , that have ( often spontaneous ) rhythmical discharge characteristics . we therefore suggest that the neuronal networks present in a spinal cord slice preparation are sufficient to generate and maintain rhythmic sympathetic activity , challenging the current view that brainstem circuits are the key contributors to the patterning of rhythmic sympathetic nerve bursts . rhythmic activity was abolished by application of ttx , indicating that neuronal firing plays a critical role in generating these rhythmic field potentials . we have also observed that iml rhythmic activity is disrupted by gap junction blockers and bicuculline . furthermore , rhythmic activity could be enhanced or induced by agonists that activate 5-ht receptors , including those selective for 5-ht2c receptors , an effect similar to that observed in vivo or in spinalized preparations . it should be noted that these network iml rhythms are a result of ensemble neuronal activity ( probably including spns and interneurons in this region ) and as such are distinct from the membrane potential fluctuations ( similarly termed  oscillations  ) in single cell recordings such as those reported in hb9-positive interneurons involved in locomotion ( wilson et al . , 2005 ) or indeed spns ( spanswick and logan , 1990 ) . however , this is not to say that these intracellular events reported by logan 's group do not correlate with , or indeed contribute to , our ensemble activity  indeed , these events are gap junction - mediated potentials ( spikelets ) ( logan et al . , 1996 ) and thus they are a probable target of our gap junction blockers . nevertheless , it is clear that our network oscillations are not simply trains of synchronized spikelets summating in the extracellular milieu  the spontaneous spikelet frequency is much too low in spinal cord slices ( mean value 1 hz ) , and spikelet frequency is increased by application of 5-ht ( pickering et al . , 1994 ) , whereas our network oscillations are increased in power by this drug but their frequency remains unaffected . it is well known that the drugs used here can have non - specific actions ( coker et al . , 2000 ; mcardle et al . , 2006 ) , such as an inhibitory effect of carbenoxolone on ca channels ( vessey et al . , 2004 ) or mefloquine on herg k channels ( traebert et al . , 2004 ) ; although with an ic50 of 2.6 m , higher than the dose used here . mefloquine and 18 -glycyrrhetinic acid can also block volume - regulated anion channels ( maertens et al . , 2000 ; ye et al . , 2009 ) and since other blockers of these channels may also have an effect at glycine receptors ( scain et al . , 2010 ) , there is a chance that these blockers may exert an action at glycine receptors . however , the fact that three different drugs exerted similar effects suggests it is their shared action at gap junctions , rather than other possible non - specific effects , which is responsible for attenuating the oscillations . this is further supported by the observation that all three gap junction blockers used disrupt gap junction - mediated spikelets in spns at these concentrations , without obvious effects on other firing properties of the neurons . although further experiments may prove unequivocally that these effects are mediated solely by an action on gap junctions , this indicates that synchronization of neuronal activity by gap junctions may be an important mechanism underpinning this rhythmic iml activity . at least some of the junctions involved may contain the subunit cx36 because mefloquine , a blocker with selectivity for cx36-containing gap junctions , replicated the effects of general gap junction blockade . a similar role of cx36-containing gap junctions in the inferior olive ( placantonakis et al . , 2006 ) , suprachiasmatic nucleus ( long et al . , 2005 ) , and hippocampus ( buhl et al . , 2003 ) has been confirmed in cx36-knockout mice . for two reasons , it is likely that the gap junctions involved in iml rhythm generation are located between spns . firstly , gap junction coupling tightly synchronizes subthreshold and suprathreshold activity between pairs of spns ( logan et al . , 1996 ) , but gap junction coupling between interneurons or between spn - interneuron pairs has not been observed . secondly , cx36 immunoreactivity has been localized to the dendritic membrane of spns , but not to interneurons , in the iml ( marina et al . , 2008 ) . in common with other cns networks ( e.g. see somogyi and klausberger , 2005 ) , spontaneous iml oscillations were also attenuated by bicuculline , a gabaa receptor antagonist , suggesting that spontaneously active gabaergic interneurons also contribute to the network rhythms . this is relevant to sympathetic control since gabaergic interneurons within the central autonomic area synapse directly onto spns ( deuchars et al . , 2005 ) and provide ongoing phasic inhibitory input in the shape of inhibitory postsynaptic potentials ( ipsps ) recorded in spinal cord slices . therefore , antagonism of the effects of these interneurons is the likely mechanism underlying the effects of bicuculline on oscillation power . these inhibitory interneurons may act to pace or reinforce the activity generated in the coupled networks , as originally observed in the hippocampus ( cobb et al . , iml rhythms were abolished by the voltage - gated na channel blocker ttx in all experiments , indicating that action potential propagation is a vital component of the oscillations . taken together with the results of gap junction and gabaa receptor blockade , these data suggest that iml field oscillations are the emergent property of a network of neurons that is dependent on synaptic properties and patterns of connections between neurons . 5-ht acts as a modulator of iml rhythmic oscillations , able both to enhance the power of existing rhythms on every occasion tested and induce rhythms in otherwise quiescent slices . this effect was mimicked by me5-ht and mk212 , suggesting this is mediated at least in part by 5-ht2 receptors . it is likely that the mechanisms underlying the spontaneous and 5-ht - induced rhythms are similar since the frequencies of spontaneous and 5-ht - induced rhythms are not significantly different and 5-ht does not significantly change the frequency of ongoing rhythms even when it enhances the power . moreover , results from the slices where cinanserin reduced the power of the oscillation provide evidence that , in these cases , at least a proportion of the spontaneous oscillations is mediated by activation of 5-ht2 receptors . where 5-ht or methyl5-ht were unable to elicit rhythmic activity in hitherto quiescent slices , it is likely that these slices were either not viable ( i.e. not healthy enough to generate complex oscillations ) , did not contain enough of the circuitry necessary for generating rhythmic activity , or may oscillate in the presence of drugs other than those of interest to us here . since the spontaneous activity level of many slices was only marginally above background noise levels , it was difficult to be certain of the viability of slices which did not display oscillations . we therefore report the existence of these slices for transparency , and with the admission that we can not say for sure whether they were the result of sub - optimal slice preparation or maintenance , or whether some may have been alive but intrinsically unable to oscillate . possible causes of the latter would include the  ladder and rung  morphology ( petras and cummings , 1972 ) of the iml , in which small clusters of spn somata are separated by highly variable gaps of 50150 m ( our unpublished estimates in 11-day - old rats ) consisting mainly of dendrites . the number of clusters per 500 m slice is therefore likely to vary , perhaps resulting in some slices containing too few spns to generate oscillatory activity . previous studies support the notion that 5-ht is an important modulator of rhythmic sympathetic activity . for example , the 10 hz rhythm in sympathetic nerve discharge is selectively enhanced by intravenous administration of a 5-ht2 receptor agonist and eliminated by a 5-ht2 antagonist , and these effects are mimicked by exciting or inhibiting serotonergic neurons in the medullary raphe respectively ( orer et al . , 1996 ) . furthermore , microinjections of 5-ht into the upper thoracic iml increased sympathetic outflow to brown adipose tissue ( madden and morrison , 2006 ) . significantly , 5-ht acting at a spinal site induces at least two distinct sympathetic rhythms ( marina et al . , 2006 ; the effects of 5-ht on iml rhythmic activity were reproduced by the selective 5-ht2c receptor agonist mk212 , suggesting that the facilitation is at least partially mediated by this receptor . indeed , 5-ht2c mrna is abundant throughout the spinal cord , including the iml ( fonseca et al . , 2001 ) . the rhythmic activity reported here was confined to the iml region of the spinal cord since , in all slices tested where spontaneous activity was present in this region , moving the electrode to other areas such as the ventral or dorsal horn resulted in loss of this activity . this suggests that , although both dorsal and ventral horn regions are capable of generating rhythmic activity if driven pharmacologically ( asghar et al . , 2005 ; nakayama et al . , 2004 ) , this particular spontaneous or 5-ht - induced rhythm is specific to the iml . it is therefore likely that it is generated locally within the iml , and represents the summed network activity of the spns and/or sympathetic interneurons in this region . the presence of rhythmic activity in the iml of a spinal cord slice strongly supports previous suggestions that rhythmic sympathetic activity can be generated by the spinal cord ( see introduction ) . the frequency of iml oscillations in the current study was in the range 7.522 hz . in vivo , the  10 hz  rhythm occurs in a similar , but much more restricted , frequency band ( 812 hz ; cohen and gootman , 1970 ; green and heffron , 1967 ) . thus , it might be speculated that the iml oscillations in the current study represent a stripped - down version of the 10 hz rhythm . in support of this idea , although it is known that at least a component of the 10 hz rhythm is generated in the brainstem , ( barman and gebber , 2000 ) , 10 hz discharges can also be generated within the spinal cord ( kubota et al . mccall and gebber , 1975 ; ootsuka et al . , 1995 ) and the 10 hz rhythm is dependent on the activity of 5-ht2 receptors ( orer et al . , however , since the closest in vivo correlate of these oscillations , the 10 hz rhythm , occurs in both young kittens and swine ( hundley et al . , 2001 ; sica et al . , 1990 ) as well as adult animals the generation of 10 hz discharges by the spinal cord is poorly understood , so this theory remains speculative . further , it will enable the contribution of individual cell types to network oscillations to be determined , since intracellular sharp electrode or patch clamp recordings may be performed in 500 m slices in conjunction with field recordings . our data demonstrate that rhythmic field potential oscillations can be recorded from the iml of neonatal rat spinal cord slices , and this activity is spontaneous in a proportion of slices . these findings suggest that the rhythmogenic capabilities of the sympathetic circuits of the spinal cord have been underestimated , and that spinal oscillators could potentially play a role in generating the physiologically - important sympathetic nerve bursts that have hitherto been credited mainly to supraspinal networks . 5-ht acting at 5-ht2 receptors modulates iml oscillations , inducing rhythmic network activity in at least a proportion of quiescent slices , and significantly enhancing spontaneous rhythmic activity in all slices tested . action potential propagation is required for the oscillations to occur , and both gap junctions ( probably between spns ) and gabaergic interneurons are important components of the spontaneous rhythmogenic network . iml oscillations in slices represent a valuable paradigm for studying the rhythmogenic capabilities of the spinal cord .