J Neurophysiol 95: 2024 –2027, 2006.
First published October 19, 2005; doi:10.1152/jn.00832.2005.
Report
Conditional Intrinsic Voltage Oscillations in Mature Vertebrate Neurons
Undergo Specific Changes in Culture
Pierre A. Guertin and Jørn Hounsgaard
Division of Neurophysiology, Department of Medical Physiology, Panum Institute, University of Copenhagen, Copenhagen N, Denmark
Submitted 8 August 2005; accepted in final form 3 October 2005
INTRODUCTION
Seminal observations first made in the invertebrate stomatogastric system have contributed to establish that intrinsic
cellular properties such as voltage oscillations can specifically
change in culture isolation presumably to maintain homeostasis
and stable activity in absence of normal synaptic events (Turrigiano et al. 1994). Generally, long-term adaptation is believed to involve modifications of intracellular calcium, gene
expression, and transcription levels that rely, in some cases, on
N-methyl-D-aspartate (NMDA) and CaV1.3 channel activation
(Bading et al. 1993). Yet, long-term changes of intrinsic (i.e.,
TTX-resistant) voltage oscillatory properties that critically depend on both NMDA and CaV1.3 channels (Guertin and
Hounsgaard 1998) have surprisingly never been examined in
mature vertebrate neurons. To investigate this question, the
present study aimed at examining the long-term effects of
culture isolation on the intrinsic oscillatory properties of mature spinal motoneurons.
METHODS
For that purpose, we used an organotypic preparation from adult
turtles developed in this laboratory that can be stably maintained for
Address for reprint requests and other correspondence: J. Hounsgaard,
Div. of Neurophysiology, Dept. of Medical Physiology, Panum Institute,
University of Copenhagen, 2200-DK, Copenhagen N, Denmark (E-mail:
[email protected]).
2024
several weeks in culture (for rational, surgical, and methological
details, see Perrier et al. 2000). In brief, 0.8- to 1.0-mm-thick transverse slices were obtained from the hindlimb enlargement spinal cord
segments (D8–S1) of adult turtles (Pseudemys scripta). Slices were
cultured at 37°C in roller tubes with culture medium for 1–3 wk and
ciliary neurotrophic factor (CNTF, n ⫽ 6), neurotrophin-3 (NT3, n ⫽
5), or glial cell line-derived neurotrophic factor (GDNF, n ⫽ 11;
Alamone, Jerusalem, Israel). GDNF and CNTF were combined in
three cases (n ⫽ 3). For acute preparations, slices were placed instead
in oxygenated physiological solution at room temperature until recording (⬍24 h). Incubated preparations consisted of acutely prepared
slices kept for 4 –24 h in oxygenated physiological solution containing
also GDNF (n ⫽ 3), CNTF (n ⫽ 3), or NT-3 (n ⫽ 1). For electrophysiological details, see Perrier et al. 2000. Oscillation frequencies
were calculated for 1 min from stable activity. Bath-applied drugs
included 10 – 80 ␮M NMDA (Tocris), 25–100 ␮M 2-amino-5-phosphonopentanoic acid (AP5, Tocris), 1–2 ␮M TTX (Sigma), 50 –100
␮M 5-hydroxytryptamine (5-HT), 10 –20 ␮M muscarine, 5–10 ␮M
BayK 8644 (RBI), and 1–10 ␮M nifedipine (Sigma). Data are presented as means ⫾ SE. Statistical significance was determined using
Student’s t-test with P ⬍ 0.05 considered as significant.
RESULTS
NMDA-induced rhythmic bursts of action potentials in most
cultured motoneurons studied (n ⫽ 18/25). In each case where
rhythmic bursting was found (Fig. 1Ai), blocking sodium
spikes with bath-applied TTX uncovered the presence of intrinsic voltage oscillations (Fig. 1Aii). We discovered that
NMDA-induced intrinsic voltage oscillations in motoneurons
were significantly (P ⬍ 0.05) faster (a 10-fold increase) in
culture than in acute preparations (Fig. 1, A vs. B). They
displayed indeed average frequencies of 1.62 ⫾ 0.24 Hz (n ⫽
18) and 0.13 ⫾ 0.01 Hz (n ⫽ 18) in culture and acute
preparations, respectively (Fig. 1D). We also found that concentrations of NMDA ⬍20 ␮M never induced motoneuronal
oscillations, whereas concentrations ⬎60 ␮M typically induced irregular and rather disorganized rhythmic depolarization in both culture and acute preparations. We noticed that
relatively high doses of NMDA were consistently required to
induce intrinsic voltage oscillations in culture (i.e., 30 ␮M in
culture vs.20 ␮M in acute preparations, see Fig. 1D). Oscillations in culture were not only faster but also significantly (P ⬍
0.05) smaller compared with those in acute preparations. Averaged amplitudes of 21.0 ⫾ 2.1 and 16.8 ⫾ 1.4 mV were
found in acute and cultured motoneurons respectively (see Fig.
1, Aii vs. B).
To examine the possibility of acute and direct modulating
effects induced by the trophic factors per se, oscillations were
The costs of publication of this article were defrayed in part by the payment
of page charges. The article must therefore be hereby marked “advertisement”
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
0022-3077/06 $8.00 Copyright © 2006 The American Physiological Society
www.jn.org
Downloaded from http://jn.physiology.org/ by 10.220.33.6 on June 16, 2017
Guertin, Pierre A. and Jørn Hounsgaard. Conditional intrinsic
voltage oscillations in mature vertebrate neurons undergo specific
changes in culture. J Neurophysiol 95: 2024 –2027, 2006. First published October 19, 2005; doi:10.1152/jn.00832.2005. Although intrinsic neuronal properties in invertebrates are well known to undergo
specific adaptive changes in culture, long-term adaptation of similar
properties in mature vertebrate neurons remain poorly understood. To
investigate this, we used an organotypic slice preparation from the
spinal cord of adult turtles maintainable for several weeks in culture
conditions. N-methyl-D-aspartate (NMDA)-induced-tetrodotoxin
(TTX)-resistant voltage oscillations in motoneurons were ⬃10 times
faster in culture than in acute preparations. Oscillations in culture
were abolished by NMDA receptor antagonists or by high extracellular Mg2⫹ concentrations. However, in contrast with results from
motoneurons in the acute slice, NMDA-induced oscillations in culture
did not depend on CaV1.3 channel activation as they still remained
after nifedipine application. Other CaV1.3 channel-mediated properties such as metabotropic receptor-induced oscillations and plateau
potentials failed to be induced in culture. This study shows that
changes specifically affecting CaV1.3 channel contribution to intrinsic
oscillatory property expression may occur in culture. The results
contribute also to understanding further the potential for plasticity of
mature vertebrate neurons.
Report
OSCILLATIONS IN CULTURED NEURONS
examined in motoneurons from acute slices incubated with
trophic factors for several hours. A representative case shown
in Fig. 1C illustrates that incubation for 7 h with GDNF (10
ng/ml) did not induce oscillations similar to those found in
culture. On average (n ⫽ 7), motoneurons incubated with
trophic factors for 4 –24 h displayed relatively slow (0.23 ⫾
0.16 Hz) and large-amplitude (21.5 ⫾ 2.3 mV) oscillations in
the presence of NMDA not significantly different (P ⫽ 0.12
and P ⫽ 0.81, respectively) from those found in acute preparations without neurotrophic factor incubation (Fig. 1B). This
demonstrates that the changes in cultured motoneurons were
not mainly induced by acute neurotrophic factor effects. In
turn, this may suggest the existence of long-term mechanisms
in response to a lack of peripheral and descending supraspinal
inputs (Turrigiano et al. 1994), although we cannot exclude the
participation to these effects of long-term and chronic actions
of neurotrophic factors on electrical properties by increasing
for instance PI3-pathway-mediated intracellular Ca2⫹ levels
and both inhibitory and glutamatergic excitatory synaptic
transmission (Arvanian et al. 2003; Perez-Garcia et al. 2004;
Schwyzer et al. 2002).
Another main finding of this study concerns a change in the
sets of calcium-permeable channels normally involved in mediating these oscillations. We found that NMDA-induced oscillations in the presence of TTX (Fig. 2i) were always abolished by AP5 (Fig. 2v, n ⫽ 4/4) and blocked by high [Mg2⫹]o
in culture (Fig. 2ii, n ⫽ 6/6). However, nifedipine (10 ␮M)
consistently failed to abolish the voltage oscillations that returned in normal [Mg2⫹]o (Fig. iii and iv). Similar results were
found in preparations that did not previously received AP5 or
other ligands (n ⫽ 7/7). Note that the applied bias currents
shown in ii and v were used mainly to ensure that the effects
were not simply the result of an increased threshold for
oscillatory property expression. To test whether CaV1.3 channels could still participate in mediating other active cellular
properties, we examined their possible implication in mediating serotonin (5-HT)-induced plateau potentials and muscarine-induced voltage oscillations. These two conditional intrinsic cellular properties are metabotropic receptor-activated and
essentially mediated by CaV1.3 channels in turtle motoneurons
from acute slice preparations (Guertin and Hounsgaard 1999;
Hounsgaard and Kiehn 1989). Interestingly, we found that
J Neurophysiol • VOL
FIG. 2. NMDA-induced voltage oscillations are independent of Cav1.3
Ca2⫹ channel activation. i–iii: 40 ␮M NMD- induced oscillations in TTX that
were reversibly blocked by high [Mg2⫹]o at 0 nA (bottom) and ⫹1.31-nA
steady bias current (top). iv: nifedipine did not change the oscillations whereas
AP5 completely abolished them at 0 nA and ⫹1.31-nA steady bias current (v).
Calibration: 10 mV.
these intrinsic properties failed to be induced in cultured
motoneurons. Plateau potentials normally expressed in acute
preparations in the presence of 5-HT after intracellular injection of a depolarizing square pulse (Fig. 3A) could not be
induced in any of the cultured motoneurons tested (Fig. 3B,
n ⫽ 6/6).
A similar lack of effect was found in the presence of BayK
8644 (n ⫽ 5/5), a selective agonist of the dihydropyridine-site
on CaV1.3 channels that normally can potently activate plateau
potential properties in motoneurons from acute preparations
(Hounsgaard and Mintz 1988). Moreover, motoneuronal voltage oscillations normally induced by muscarine in acute preparations failed to be induced in culture (n ⫽ 4/4, not shown).
FIG. 3. Serotonin (5-HT)-induced effects on plateau potentials and oscillations. A: in an acute motoneuron, an intrasomatic depolarizing 0.8-nA current
pulse induced a self-terminating plateau potential in the presence of 10 ␮M
5-HT. B: depolarizing current pulse did not induce plateau potential in a
cultured motoneuron. C: rhythmic activity induced by 40 ␮M NMDA (i) that
became intrinsic oscillations in TTX (ii) remained still with 100 ␮M 5-HT (iii).
95 • MARCH 2006 •
www.jn.org
Downloaded from http://jn.physiology.org/ by 10.220.33.6 on June 16, 2017
FIG. 1. N-methyl-D-aspartate (NMDA)-induced rhythmicity. A: motoneuron cultured with 100 pg/ml GDNF for 10 days displayed NMDA-induced
activity (i) that became oscillations after TTX application (ii). B: acute
motoneuron displaying oscillations induced by 30 ␮M NMDA and 1 ␮M TTX.
C: 30 ␮M NMDA-induced oscillations in a motoneuron incubated with 10
ng/ml GDNF for 7 h. D: frequency of NMDA-induced oscillations plotted
against NMDA concentrations ranging from 20 to 80 ␮M.
2025
Report
2026
P. A. GUERTIN AND J. HOUNSGAARD
DISCUSSION
These results show that NMDA generally induced fast TTXresistant voltage oscillations in organotypic cultured motoneurons. These were insensitive to nifedipine but blocked by AP5
or high [Mg2⫹]o. Muscarine, 5-HT, and BayK 8644 were
found not to uncover or modulate oscillations and plateau
potentials. Otherwise, action potential, resting potential, and
input resistance values remained relatively unchanged in organotypic cultures and acute preparations.
Voltage oscillations induced by NMDA were also abnormally fast as they were ⬃10 times faster in culture (1.62 Hz)
than in acute preparations (0.13 Hz, Fig. 1D). The high frequencies found in culture are, in fact, similar to those reported
in acute preparations from neonatal preparations (i.e., 1–3 Hz)
(Hochman et al. 1994; MacLean et al. 1998). Yet it is unlikely
that the motoneuronal oscillation frequencies found in acute
preparations were atypically slow or specific to turtles because
comparable NMDA-induced frequencies have been reported in
other adult vertebrate species (i.e., ⬍0.3 Hz in rat, Durand
1991; in lampreys, Wallén and Grillner 1987).
Some of the changes in neuronal response properties during
culture deviate from previous studies. For example, while we
found that voltage oscillations in motoneurons are transformed
in culture (i.e., faster frequencies and fewer activating systems), the ability to generate CaV1.3-dependent plateau potentials is completely abolished (Fig. 3B) (see also Perrier et al.
2000). Neurons in the lobster stomatogastric ganglion system,
which normally express tonic depolarization in acute preparations, acquire oscillatory properties as an intrinsic property and
become unconditional bursters after a few days in culture
(Turrigiano et al. 1994). Also in contrast with our results, the
bursting activity that is expressed in embryonic neurons (not
identified as motoneurons) is not intrinsic (i.e., not TTXresistant) but depends on network activity in culture (Ballerini
et al. 1999; Keefer et al. 2001; Streit 1993). Altogether, this
J Neurophysiol • VOL
may indicate that intrinsic properties of mature vertebrate
neurons adapt differently to culture conditions than invertebrate neurons and neonatal vertebrate neurons. Reasons for this
are unclear, but given the differences known to exist between
some of these systems (e.g., neonates vs. adults), developmentally mature systems could undergo some regression associated
with a return to non-fully functional CaV1.3 channels as in
neonates (Jiang et al. 1999). This hypothesis has been proposed
already by Perrier and colleagues who have shown a complete
loss of CaV1.3-dependent plateau potential properties in a
similar preparation (Perrier et al. 2000).
Such possible down-regulation of CaV1.3 channels is supported by data showing that nifedipine (i.e., as high as 10 ␮M)
consistently failed to abolish the fast oscillations induced by
NMDA in cultured motoneurons (Fig. 2). Given that nifedipine
generally completely blocks NMDA-induced oscillations in
acute preparations (Guertin and Hounsgaard 1998), this
strongly support the idea that CaV1.3 channels lost their
capacity to mediate this property in culture. The idea is further
supported by the fact that direct channel activation with BayK
8644, a highly potent agonist for CaV1.3 channels, did not
induce plateau potentials, as it does normally in acute slice
preparations (Hounsgaard and Kiehn 1989). Given that the
plateau potential in turtle motoneurons is an intrinsic property
essentially mediated by CaV1.3 channels (Hounsgaard and
Kiehn 1989; Simon et al. 2003), this suggests, again, that
CaV1.3 channels were functionally down-regulated. This could
be the result of reduced CaV1.3 channel transcript levels in
culture as reported with cultured Purkinje neurons (Gruol et al.
1992).
On the other hand, NMDA ionophores were shown to
remain a key factor in mediating oscillations in cultured motoneurons as they are in acute preparations (Guertin and Hounsgaard 1998). We showed, in fact, that the NMDA receptorchannel system remained critically important because oscillations in culture were completely blocked by AP5 or high
[Mg2⫹]o (Fig. 2). In turn, this may suggest the existence of a
compensatory mechanism by which the NMDA ionophore had
to increase its ionic conductance level or that a small undetectable increase of its density resulted in greater calcium entry
(Turrigiano et al. 1995) to allow voltage oscillations to still be
inducible despite a presumably down-regulated CaV1.3 channel system.
As mentioned earlier, we found that 5-HT lost its capacity to
modulate NMDA-induced voltage oscillations (Fig. 3C) and to
uncover plateau potentials (Fig. 3B). We reported also that
muscarine consistently failed to induce or modulate voltage
oscillations in culture. Because in acute preparations, 5-HT and
muscarine can normally induce and modulate these properties
(Guertin and Hounsgaard 1999), the present results show that
the 5-HT and muscarine receptor systems lost their capacity to
affect these properties in culture. This loss of functions may be
explained by downstream changes affecting second-messenger
systems or CaV1.3 channel function as mentioned earlier.
Because NMDA-induced oscillations in culture were not modulated by muscarine or by 5-HT (Fig. 3C) as they should
normally via functionally-coupled intracellular signaling systems between NMDA ionophores and 5-HT receptors for
instance (MacLean and Schmidt 2001; MacLean et al. 1998),
this could argue also for a down-regulation of metabotropic
95 • MARCH 2006 •
www.jn.org
Downloaded from http://jn.physiology.org/ by 10.220.33.6 on June 16, 2017
Finally, NMDA-induced oscillations in cultured motoneurons
were not affected by bath-applied muscarine or 5-HT. This is
shown in Fig. 3C where NMDA-induced bursts (i) transformed
by TTX into intrinsic voltage oscillations (ii) remained of
relatively large amplitude and slow frequency after addition of
5-HT (iii). On average (n ⫽ 6), oscillation frequencies and
amplitudes (1.72 ⫾ 0.20 and 18.1 ⫾ 1.5) were not significantly
(P ⬎ 0.05) changed after 5-HT or muscarine application
(1.58 ⫾ 0.31 and 15.7 ⫾ 2.1). further supporting the idea that
metabotropic receptor activation with 5-HT or muscarine did
not affect the channels under these conditions.
Otherwise, impaled motoneurons from this organotypic culture preparation (n ⫽ 18) displayed action potentials, resting
membrane potentials and input resistance values that were not
significantly different (P ⬎ 0.05) compared with those normally found in acute preparations (n ⫽ 18). The average
peak-to-peak action potential amplitude was smaller, although
not significantly (P ⫽ 0.07), in culture compared with acute
preparations (53.0 ⫾ 3.1 vs. 63.9 ⫾ 2.6 mV). However, the
input resistance and resting membrane potential values of
cultured motoneurons were of 18.1 ⫾ 1.7 M⍀ and ⫺57.4 ⫾
1.5 mV, which are not significantly different (P ⬎ 0.05)
compared with those found in motoneurons from acute preparations (16.8 ⫾ 0.9 M⍀ and ⫺62.6 ⫾ 1.2 mV, respectively).
Report
OSCILLATIONS IN CULTURED NEURONS
receptor system-activated intracellular pathways or of the 5-HT
and muscarine receptors themselves.
ACKNOWLEDGMENTS
Thanks to H. Jahnsen, J. F. Perrier, and I. Kjaer for help with cultures.
GRANTS
This work was supported by Medical Research Council Canada, Medical
Research Council Denmark, and The Lundbeck Foundation.
REFERENCES
J Neurophysiol • VOL
Hounsgaard J and Mintz I. Calcium conductance and firing properties of
spinal motoneurons in the turtle. J Physiol 398: 591– 603, 1988.
Jiang Z, Rempel J, Li J, Sawchuk MA, Carlin KP, and Brownstone RM.
Development of L-type calcium channels and a nifedipine-sensitive motor
activity in the postnatal mouse spinal cord. Eur J Neurosci 11: 3481–3487,
1999.
Keefer E, Gramowski A, and Gross GW. NMDA receptor-dependent periodic oscillations in cultured spinal cord networks. J Neurophysiol 86:
3030 –3042, 2001.
MacLean JN, Cowley KC, and Schmidt BJ. NMDA receptor-mediated
oscillatory activity in the neonatal rat spinal cord is serotonin dependent.
J Neurophysiol 79: 2804 –2808, 1998.
MacLean JN and Schmidt BJ. Voltage-sensitivity of motoneuron NMDA
receptor channels is modulated by serotonin in the neonatal rat spinal cord.
J Neurophysiol 86: 1131–1138, 2001.
Perez-Garcia MJ, Cena V, de Pablo Y, Llovera M, Comella JX, and Soler
RM. Glial cell line-derived neurotrophic factor increases intracellular calcium concentration. Role of calcium/calmodulin in the activation of the
phosphatidylinositol 3-kinase pathway. J Biol Chem 279: 6132– 6142, 2004.
Perrier J-F, Noraberg J, Simon M, and Hounsgaard J. Dedifferentiation of
intrinsic response properties of motoneurons in organotypic cultures of the
spinal cord of the adult turtle. Eur J Neurosci 12: 2397–2404, 2000.
Schwyzer L, Mateos JM, Abegg M, Rietschin L, Heeb L, Thompson SM,
Luthi A, Gahwiler BH, and McKinney RA. Physiological and morphological plasticity induced by chronic treatment with NT-3 or NT-4/5 in
hippocampal slice cultures. Eur J Neurosci 16 :1939 –1948, 2002.
Simon M, Perrier JF, and Hounsgaard J. Subcellular distribution of L-type
Ca2⫹ channels responsible for plateau potentials in motoneurons from the
lumbar spinal cord of the turtle. Eur J Neurosci 18: 258 –266, 2003.
Turrigiano GG, Abbott LF, and Marder E. Activity-dependent changes in
the intrinsic properties of culture neurons. Science 264: 974 –977, 1994.
Turrigiano GG, LeMasson G, and Marder E. Selective regulation of current
densities underlies spontaneous changes in the activity of cultured neurons.
J Neurosci 15: 3640 –3652, 1995.
Streit J. Regular oscillations of synaptic activity in spinal networks in vitro.
J Neurophysiol 70: 871– 878, 1993.
Wallén P and Grillner S. N-methyl-D-aspartate receptor-induced, inherent
oscillatory activity in neurons active during fictive locomotion in the
lamprey. J Neurosci 7: 2745–2755, 1987.
95 • MARCH 2006 •
www.jn.org
Downloaded from http://jn.physiology.org/ by 10.220.33.6 on June 16, 2017
Arvanian VL, Horner PJ, Gage FH, and Mendell LM. Chronic neurotrophin-3 strengthens synaptic connections to motoneurons in the neonatal rat.
J Neurosci 23 :8706 – 8712, 2003.
Bading H, Ginty DD, and Greenberg ME. Regulation of gene expression in
hippocampal neurons by distinct calcium signalling pathways. Science 260:
181–186, 1993.
Ballerini L, Galante M, Grandolfo M, and Nistri A. Generation of rhythmic
patterns of activity by ventral interneurons in rat organotypic spinal slice
culture. J Physiol 517: 459 – 475, 1999.
Durand J. NMDA actions on rat abducens motoneurons. Eur J Neurosci 3:
621– 633, 1991.
Gruol DL, Deal CR, and Yool AJ. Developmental changes in calcium
conductances contribute to the physiological maturation of cerebellar Purkinje neurons in culture. J Neurosci 12: 2838 –2848, 1992.
Guertin PA and Hounsgaard J. NMDA-induced intrinsic voltage oscillations
depend on L-type calcium channels in spinal motoneurons of adult turtles.
J Neurophysiol 80: 3380 –3382, 1998.
Guertin PA and Hounsgaard J. L-type calcium channels but not N-methylD-aspartate receptor channels mediate rhythmic activity induced by cholinergic agonist in motoneurons from turtle spinal cord slices. Neurosci Lett 26:
81– 84, 1999.
Hochman S, Jordan LM, and Schmidt BJ. TTX-resistant NMDA receptormediated voltage oscillations in mammalian lumbar motoneurons. J Neurophysiol 72: 2559 –2562, 1994.
Hounsgaard J and Kiehn O. Serotonin-induced bistability of turtle motoneurons caused by a nifedipine-sensitive calcium plateau potential. J Physiol
414: 265–282, 1989.
2027