Whole cell voltage clamp recordings from cultured neurons of the supraoptic area of neonatal rat hypothalamus

Brain Research, 409 (1987) 175-180 Elsewer

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Whole cell voltage clamp recordings from cultured neurons of the supraoptic area of neonatal rat hypothalamus Peter Cobbett and Wllham T Mason Department of Neuroendocrmologv AFRC Instttute of Antmal Phystologv and Genet:cs, Babraham 6ambnd~e ( U lx )

(Accepted 6 January 1987) Key words Supraoptic neuron, Cell culture, Voltage clamp, Na-current, Ba-current

Whole-cell voltage and current clamp recordings were made from cultured neurons from the hypothalam~csupraopt~c area of neonatal rats These neurons fired spontaneous Na+-actlon potentials, appearing as mixed Na+/K+-currents in voltage clamp Isolated Na+-currents (< 3 nA) were rapidly activated and reactivated during posltlve potential pulses from -80 mV Two voltage-acnvated Ba2+-currents (< 1 nA) were also recorded These techniques offer a promising new approach for studying the striking electrical behavior of cultured hypothalamxcneurons

The magnocellular oxytocln and vasopressln neurons which comprise the hypothalamoneurohypophysml system in mammals exhibit characteristic brIng patterns in wvo u n d e r certain conditions Speclhcally, ox~tocln neurons hre a high-frequency dis-

tlals are superimposed oscillations of the m e m b r a n e potential However other features of phasic neurons may also be recorded lrom neurons (putative oxytocinerglc) that do not fire repetltwe bursts of action potentmls but exhibit many of the same leatures as

charge just prior to each milk ejection in lactating an-

the phasic neurons For example, a Ca :+ component

imals, whereas vasopressln neurons fire phasic bursts of action potentmls during osmotic or hemorrhagic stimuh (see ref 21 for review) To understand the mechanisms underlying these

to the action potentml :~ ta a depolansmg afterpotentlal following the action potential 1-3 7 a sustained

different firing patterns and to determine whether oxytocln and ~asopressln neurons have different m e m b r a n e properties, conventional mtracellular ~oltage recordings have been made from these neurons m various in wtro preparations These are (1) from adult rats slices containing supraoptlc (SON) and p a r a v e n t n c u l a r (PVN) nuclei I-5 13 1920 and perfused explants containing the SON 7-9, (2) organotyplc cultures of neonatal rat hypothalamus l~ 16, and (3) dispersed cell cultures of fetal mouse hypothalamus~ ,_2 In all these in v~tro p r e p a r a n o n s some neurons f|re spontaneous phasic bursts ot action potentials or ma'~ fire such repetlt~ve bursts during Injection ot 1o~ mtenslt 3 current bursts of action poten-

burst of action potentials following an appropriate stimulus ~,- and prolongation of the action potentml with increased firing rates ~ ~ ,-f~ To determine the m e m b r a n e p r o p e m e s of these neurons v~e have made ~oltage and current clamp recordings from cultured neurons and report here some of the p r o p e m e s ot currents flowing through voltage activated Na +- and Ca:*-channels All recordings were made from neurons m cultures 1-3 weeks old Cultures were prepared from the supraoptlc nuclei and the immediately surrounding area of -2 to 4-day-old rats, (each piece ol tissue was about 1 mm x 1 5 r a m × 1 5 mm) P r o c e d u r e s ~ d l be detailed elsewhere and thus wdl onl~ be brlefl,¢ described here After decapitation and removal of the brain all subsequent procedures were performed

(orrewondenee P Cobbett Department ol Neuroendocrmolog,~ AFRC Insutute of Ammal Phvslolog,~ ,rod Genetic, Babraham CambndgeCB24Aq U K

0006-899~ 87,$(B S()© 1987 ENevler Science Pubh~hers B ¥ (Biomedical Dwlslon)

176 using standard sterile tissue culture techmques Tissue pieces from 8 animals were gently agitated in papain solution (Sigma, 0 5 mg/ml in phosphate-buffered saline) at 37 °C Agitation was briefly Interrupted to triturate the tissue through two fire polished pipettes To the resultant cell suspension, an equal volume of culture medium (Dulbecco's modified Eagle's medium supplemented with 25% newborn calf serum) was added, and after mixing, the cell suspension was filtered and centrifuged The pellet was resuspended in 1-2 ml of culture medium and cells were plated out onto petrl dishes (Nunc, 35 mm) For recordings, dishes were mounted on the stage of an inverted microscope (Leltz Dlavert) The culture medium was replaced by solution containing (in mM) (1) NaC1 125, HEPES 10 (pH 7 3), glucose 5 56, and tetraethylamonlum Br 20 for recording pure Na+-currents, or (2) NaCl 140, MgCl 2, glucose 10, KCI 2, and H E P E S 10 (pH 7 3) for recording mixed currents and membrane potentials, or (3) Choline C1 145, HEPES 10 (pH 7 3) glucose 5 56, 10 mM BaCl 2 for recording isolated barium currents Whole cell recordings were made using the glgaseal technique originally described by HamlU et al 16 with electrodes containing (m mM) either KCI 140, MgCl 2 2, E G T A 1, and H E P E S 10 (pH 7 3) to record mixed currents and membrane potentials, or CsC1 140, E G T A 5, HEPES 10 (pH 7 3), MgCI 2, and glucose 10 for recording isolated Na +- and Ba2+-cur rents All recordings were made with optimal compensation (70-90%) of the series resistance so that the maximum voltage drop was 4 mV Signals were amplified (List EPC 7) and stored on magnetic tape for subsequent computer (DEC PDP 11/73) assisted analysis Data acquisition, current averaging, subtraction of capacltatlve and/or linear leakage curents, and final display ot records were performed using a suite of programs written by Dr T D Lamb (University of Cambridge, U K ) Neurons were recognlsable as having a phase bright cell body with 1 to 4 processes (Fig 1) Immunocytochemlcal staining demonstrated that cells morphologically similar to those from which records were obtained were reactive to anti-vasopressm or antl-oxytocln sera (A detailed description ol the morphology and immunocytochemlstry of these cells will appear elsewhere )

Fig 1 Examples of phase bright neuron, (arrows) of the tvpe from which recordings were made m t0-da~-old cultures obtained from the supraoptlc area of 2-da~-otd rats Bar = 25 um

Under voltage clamp, with internal K + and external Na + and K +, positive potential pulses from a holding potential of - 8 0 mV produced a current with an early Inward component and a delayed outward component (Fig 2A) The reward, constant latency component of the somatic current had a threshold of about -55 mV, (low threshold inward currents with variable latency trom the pulse onset were recorded in some instances and these were interpreted as being dendrmc in origin) Usually the current was maximal 10- t5 mV beyond threshold at more posmve potentials, the inward current decreased as the delayed outward K+-current was more rapidly activated and the pulse potential approached the reversal potential for Na + Under current clamp, the cells had resting potentials o f - 4 5 mV to -80 mV, fired spontaneous action potentials having a threshold ot about -60 mV and

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Fig 2 Voltage and current clamp recordings from the same neuron A voltage clamp recordings of mixed Na+/K+-currents in response to positive voltage pulses from the holding potential, (the outward current responses to the largest potential jumps are not shown) A low threshold, variable latency inward Na+-current was recorded from this cell (arrow) Each record (except the low threshold response) is an average of 5 responses, with capacltatlve currents subtracted (the data was filtered at 2 kHz and dlgitlsed at 8 kHz) B current clamp records of the potential response to pulse injection of current of various amplitudes (Data was digltlSed at 2 kHz) C continuous record showing spontaneous action potentials some of which appear to be generated by a post synaptic potential (arrows), the resting potential of this neuron was -57 mV

had input resistances of more than 1 Gf2 (Fig 2B) The action potentials overshot 0 inV. with the membrane potential being briefly positive, had a prominent afterhyperpolar]satlon, and were 1 - 4 ms m duration Spontaneous firing rates varied (Fig 2C), but when the potential was held just below the threshold, non-reactivating action potentials could be sustained at frequencies up to 20 Hz Action potent]Ms and the reward c o m p o n e n t of the mixed current were blocked by tetrodotoxln (TTX, 1-5/~M) to the external m e d m m A d d m o n of Ca 2+ ( 5 - 1 0 mM) to the dzsh produced a prolongation of the spike duration of less than 10%

Th]s unexpected result could be attributed to (a) the presence of a voltage activated Ca2+-channel which IS only slowly activated, and/or (b) the presence ot relatively rapidly activating K+-currents that obscure the effects of Ca2+-channel activation The last can be considered a distract possibility since even in the absence ot Ca 2+ the action potential has a strong atterhyperpolarlsatlon To examine such possibilities m more detail, Na + and Ba2+-currents were recorded m ~solat~on For these experiments internal K + was subsututed by Cs +, and the external medium contained either Na + or Ba 2+ as the charge carrying species Low-

178 tentmls but maximal at a test p o t e n h a l lmmedJatel~ suprathreshold which would lmpl~ a ~teep slope for the actwat~on of the current duc to poor spatml clamp In most neurons, the ~.urrenl was ot constant latency at each potenual throughout the range tested The current was rapidly actwated and lnactl-

threshold tsolated Na+-currents (-65 mV) w~th varmble latency from pulse onset were recorded from some cells these were mtepreted as being dendrmc m origin and signify that the spatial clamp of the cell was not ophmal We excluded all data from recordings m which the current was zero at subthreshold po-

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Fig 3 Voltage clamp recordings of isolated Na +- and Ba2+-currents A isolated Na+-currents recorded at various potentmls (as mdzcated) durmg voltage pulses from a holding potennal of -90 mV (Data are average of 5 responses, with capamtatlve currents subtracted, and was filtered at 2 kHz and digmsed at 8 kHz ) B rsolated Ba 2+-currents recorded at various potenuals (as indicated holdmg potential -80 mV) Each record is an average of 5 responses, with hnear leakage currents subtracted, and data was digmsed at 4 kHz and filtered at 1 kHz Note that actwat~on ~s much slower than for the Na+-currents and that mactavalaon Is incomplete C. roachvat,on of isolated Na+-current during pulse trams of different frequencies Each response Is expressed as a fraction of the maximum response obtained during that tram, ~ e fractton non-mactwated current, (5 pulses per tram, 30 s between consecunve trams) tnactwat~on ~s frequency dependent, but is less than 30% even at 20 Hz D current-voltage relanonshtp for the lsolatedcurrents dlustrated m A and B The membrane current (/.1) ~s plotted m relanve umts wtth absolute maximum values gwen Note that for the Ba2+-current the peak and end (of 150-ms pulse) values are plotted

179 vated so that it reached its peak within 1 ms ot pulse onset and decayed to a steady-state level within 5 ms of the pulse onset (Fig 3A) F r o m threshold (-55 8 +_ 1 5 m V , m e a n +_ S E M , n -- 12), the p e a k current ~ a s - I 92 _4- 0 2 n A and occurred +12 1 ± 0 9 m V t o the threshold The whole cell conductance was calculated to be 19 8 ± 5 7 nS, and the reversal potential was calculated to be 49 5 ± 4 6 mV (Fig 3D, actual reversal o1 the current was not observed) The Na +current was completely blocked by T T X ( 2 - 5 / ~ M ) As expected from the ability, ot these cells to fire Na+-actlon potentials at high lrequencles without signihcant decrease in amplitude, the Na+ current was usually less than 20% inactivated at frequencies up to 40 Hz (Fig 3C) The voltage-activated Ca:+-channels were examlned using Ba 2+ as the charge carrier These currents were o~ a completely different nature to the isolated Na+-current T ~ o c o m p o n e n t s to the total current a p p e a r e d to be present in some neurons These were, /lrstl~ a low threshold ( - 4 5 7 + l 8 mV, n = 12) "rapidly' activating and Inactivating c o m p o n e n t and secondly, a higher threshold c o m p o n e n t that was inactivated little during the course ot a voltage pulse lasting 150 ms (Fig 3B) These two c o m p o n e n t s represent two independently operating populations ot C a > - c h a n n e l and not a complex multlstate single channel since in some cells the c u r r e n t - v o l t a g e ( l - V ) relationship showed two distinct peaks (Fig 3D) These two populations may be distributed Slmllarl~ over the whole m e m b r a n e and have different kinetics etc or may be differentially distributed (between soma and dendrites) In other cells, however only the slowly inactivating current a p p e a r e d to be present Taking all the cells as a single population, we tound maximal currents to be - 5 4 8 4 ± 79 6 p A which occurred at +30 0 + 3 3 mV to the threshold The linear portxon of the I - V relationship gave a whole cell conductance o1 9 2 + 1 4 nS and a reversal potential ot 47 5 ± 2 9 m V These Ba~+-currents were unaffected by TTX at concentrations that totails suppressed Na+-currents, and were blocked by other divalent cations such as Co :+ and Mn -~+ (2-5 mM) This IS the first r e p o r t of electrophysiologlcal recordings from dispersed neonatal hypothalamlc neurons Clearl~¢ the neurons are viable in culture and retain morphological and electrophyslologlcal

features ot neurons in SltU In particular, these cells are refractlle under phase contrast optics and the ~oltage-actlvated Na+-currents or action potentials may be sustained at high frequencies In contrast, putative gllal cells were fiat in culture, and previous recordings lorm gila indicate that although Na+-cur rents mav be voltage activated the}' are ot ~er,, different nature 6 One leature of Na+-currents In these neurons was that the potentials ot current threshold and tor maximum current activation were relatively negative and s e p a r a t e d b~ less than 15 mV This is in contrast to other excitable cells m which these currents are maximal up to 41) mV positive to the threshold ~ I~ l_, That we were unable to record large changes in the shape of the action potential following addition oI calcium to the external m e d i u m is of some interest C l e a r b these neurons possess voltage activated C a > - c h a n n e l s , as we have d e m o n s t r a t e d here, but their activation is relatively slow Further it is possible that voltage activated K+-currents are rapidly activated and thus prevent the a p p e a r a n c e ol the Ca :+component or shoulder' ot the action potential which ma1~ be recorded trom adult supraoptic neurons m vltroSS is > The nature ot the potassium c u r r e n t b ) m these neurons has not been studied yet and only alter such a study and examination ot K + and Ca z+currents in older cultures will it be possible to determine v~h) these (immature) neurons do not display broad action potentials A n o t h e r feature of possible lmmaturlt} of these neurons ma~ be the presence ot two components of the voltage-activated Ca2+-currents The low-threshold "rapidly' inactivating c o m p o n e n t has only been described in spinal cord and dorsal root ganglion neurons in culture which have been obtained trom neonatal mammals t4 and from embryonic chick neurons m However It is also possible that these supraoptic neurons normally possess two CaZ+-channels and that these ma) be a part of the mechanism controlling phasic tiring in vasopresslnerglc neurons In conclusion we have d e m o n s t r a t e d the viability ot neonatal neurons of the supraoptlc area ot the hypothalamus in dissociated culture and have m a d e whole-cell voltage and current clamp recordings from these cells This work has shown that the cultured cells and possess voltage-activated ion channels postulated to be present In adult neurons This

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