two distinct types of repetitive bursting activity mediated by nmda in hypothalamic neurons in vitro

Two distinct types of repetitive bursting activity mediatedby NMDA in hypothalamic neurons in vitro

Pierre PoulainU422, INSERM, Place de Verdun, 59045 Lille Cedex, France

Keywords: guinea pig, low-threshold spike, slice, whole-cell

Abstract

Hypothalamic magnocellular dorsal nucleus neurons were recorded from adult guinea pig brain slices with the whole-cell patch-clamp technique to determine the effects of N-methyl-D-aspartate (NMDA) applied in the bath or by iontophoresis. In a majority of

cells (59 of 77, 76.6%), rhythmic bursting discharges were evoked by speci®c activation of NMDA receptors when the membrane

was more negative than ±60 mV. This endogenous rhythmic activity was resistant to tetrodotoxin. It was suppressed by removalof extracellular Mg2+, indicating the involvement of the voltage-dependent block of the NMDA channel by Mg2+. Application of

thapsigargin showed that rhythmic activity did not depend on the release of Ca2+ from reticulum stores. Blockers of Ca2+

conductances Ni2+ and nifedipine had no effects on the bursts. Their repolarization did not involve the activation of a

strophantidin- or ouabain-sensitive pump, but partly depended on an apamine-sensitive Ca2+-dependent K+current. In a smallsubset of cells (9 of 69, 13%), speci®c activation of NMDA receptors induced another type of bursting activity which consisted of

repetitive low-threshold spikes sustaining bursts of action potentials. Rhythmic low-threshold spikes subsisted in the presence of

tetrodotoxin but were suppressed by Ni2+. Increasing the amount of NMDA brought about a switch from the rhythmic low-threshold spike burst ®ring to the rhythmic bursting activity observed for the majority of cells. The present data show for the ®rst

time that NMDA receptor activation can induce two independent rhythmic bursting behaviours in the same neuron, probably

depending on the strength of the glutamatergic drive.

Introduction

Rhythmic membrane depolarizations and associated bursts of

action potentials depending upon the speci®c activation of N-

methyl-D-aspartate (NMDA) receptors have been observed in a

variety of neurons submitted to application of NMDA. On the one

hand, they have been observed in motoneurons of lower

vertebrates (Wallen & Grillner, 1987; el Manira et al., 1994;

Sillar & Simmers, 1994; Rioult-Pedotti, 1997; Guertin &

Hounsgaard, 1998; Prime et al., 1999) and rat (Durand, 1993;

Hochman et al., 1994; Kim & Chandler, 1995; MacLean et al.,

1998; Schmidt et al., 1998) and in neurons of mammalian

brainstem (Johnson et al., 1992; Sera®n et al., 1992; Tell & Jean,

1993). In these neurons, rhythmic bursting discharges induced by

NMDA receptor activation may be implicated in rhythmical motor

behaviours. On the other hand, NMDA-induced rhythmic bursts

have also been recorded from populations of mammalian neurons

located in diverse brain territories, and then are involved in

various functions (Flatman et al., 1986: sensorimotor cortex; Hu

& Bourque, 1992: hypothalamus; Meier & Herrling, 1993:

habenula; Bacci et al., 1999: hippocampus). In all these obser-

vations, NMDA-induced rhythmic bursting discharges have in

common that they involve intrinsic membrane properties and

require the presence of physiological Mg2+. As a rule, suppression

of the negative slope region of the NMDA channel's current±

voltage relationship (Nowak et al., 1984; Flatman et al., 1986),

via removal of Mg2+, suppresses rhythmic bursts. Nevertheless,

even if the patterns of the Mg2+-dependent rhythmic bursting

discharges are virtually the same in the different preparations,

rhythmicity appears to arise through a variety of cellular

mechanisms.

Another type of repetitive bursting activity has been recorded

during application of NMDA to mammalian neurons able to display

low-threshold spikes (LTS) due to T-type Ca2+ currents (Leresche

et al., 1991; Khateb et al., 1995, 1997) This unusual pattern of

rhythmicity, although induced by activation of NMDA receptors, is

independent of extracellular Mg2+. In this case, rhythmicity depends

upon the sustained repetition of individual LTS, each of them giving

rise to a burst of action potentials. The coexistence in the same

neuron of two different NMDA-induced patterns of rhythmicity,

Mg2+-dependent rhythmic bursting discharges and repetitive LTS

discharges, has not yet been observed. Furthermore, a possible

involvement of T-type currents in the Mg2+-dependent rhythmic

bursting discharges is unknown. To address these questions, the

guinea pig magnocellular dorsal nucleus (MDN) neurons are an

attractive model because they are endowed with a powerful LTS

(Niespodziany & Poulain, 1995). Properties of the T-type current

have been recently investigated in these neurons (Niespodziany et al.,

1999). In the present study, during whole-cell patch-clamp recordings

of MDN neurons from fresh slices, NMDA was observed to trigger a

repetitive bursting activity. Thus, the objectives were, ®rst, to study

the mechanisms underlying the discharge induced by NMDA in order

to compare them with those recorded in other preparations and,

second, to test the contribution of the T-type current in the generation

of the discharges.

Correspondence: Dr Pierre Poulain, as above.E-mail: [email protected]

Received 13 December 2000, revised 21 May 2001, accepted 18 June 2001

European Journal of Neuroscience, Vol. 14, pp. 657±665, 2001 ã Federation of European Neuroscience Societies

Materials and methods

Brain slices were prepared from female guinea pigs weighing 125±

250 g. The animal was killed by decapitation, the brain was quickly

removed and immersed in cold (1±4 °C), oxygenated (95% O2/5%

CO2), arti®cial cerebrospinal ¯uid (ACSF). The composition of the

ACSF was (in mM): NaCl, 120; KCl, 5; CaCl2, 2; MgCl2, 1.2;

NaHCO3, 25; NaH2PO4, 1.2; and glucose, 10 (pH 7.4 with 95% O2/

5% CO2, osmolarity 300±320 mosmol/kg). All compounds were

purchased from Sigma Chemicals (Saint Quentin Fallavier, France)

unless otherwise stated. Two consecutive slices containing the MDN,

400 mm thick, were cut transversely with a Vibroslice (Campden

Instruments, Leicester, UK) and transferred to an interface-type

recording chamber. The slices were thermoregulated (32±34 °C),

oxygenated (95% O2/5% CO2), and superfused (2 mL/min) with

ACSF. Slices were allowed to equilibrate for approximately 1 h

before starting recordings.

Current-clamp recordings were performed using the whole-cell

con®guration of the blind patch-clamp method. Signals were recorded

in the bridge mode using an Axoclamp 2A (Axon Instruments, Foster

City, CA, USA), displayed on an oscilloscope (Gould 1604, Les Ulis,

France), and stored on a digital tape recorder (DTR 1201, Biologic,

Claix, France). Patch pipettes (2 mm tip diameter, 5±10 MW) were

®lled with a solution containing (in mM): K+ gluconate 130; MgCl2,

1; CaCl2, 1; Hepes, 10; ATP-Mg, 2; and EGTA, 10 (pH adjusted to

7.2 with KOH, osmolarity of 280±295 mosm/kg). Biocytine (2%)

was added to the pipette solutions to label the neurons. Liquid

junction potentials between the pipette solution and the ACSF were

measured and corrected accordingly.

Drugs were dissolved in ACSF and applied through the perfusion

medium. They included: apamin, DL-2-amino-5-phosphonopentanoic

acid (AP5), L-glutamate, iberiotoxin (Alomones Laboratories,

Jerusalem, Israel), nickel chlorure, NMDA, ouabain, strophanthidin,

tetrodotoxin (TTX; Alomones Laboratories) and thapsigargin.

Nifedipine was ®rst dissolved in ethanol absolute and then diluted

in the normal ACSF with a ®nal concentration of alcohol of 0.01%.

To test for Ca2+ selectivity, equimolar amounts of MgCl2 were

substituted for CaCl2 in the ACSF. NMDA (30±100 mM in distilled

water) was also applied by iontophoresis with micropipettes made

with two-barrelled theta-style capillary tubing (Clark Electromedical

Instruments, Edenbridge, UK), 2 mm o.d. Tips of the pulled

micropipettes were 4 mm in diameter. Negative currents were

programmed by a Neurophore (Digitimer, Hertfordshire, UK) to

eject NMDA through one barrel, whereas the other barrel was ®lled

with ACSF for an automatic balancing of the ejection current.

After recording, the slice was ®xed in picric acid±paraformalde-

hyde overnight. It was then left for 1 h in phosphate buffer 0.1 M,

pH 7.4, containing 20% sucrose, before it was embedded in Tissue-

Tek (Sakura, Zoetewoude, The Netherlands) and frozen. Sections,

30 mm in thickness, were cut on a cryostat, mounted on gelatin-

coated slides and stocked at ±80 °C. Before revealing biocytin,

sections were rehydrated in phosphate buffer and incubated in a

solution containing 20% methanol and 5% H2O2 (33%) in phosphate

buffer for 15 min to suppress endogenous peroxidase. After washing,

sections were treated with Vectastain Elite ABC kit according to the

recommendations of the manufacturer (Vector Laboratories,

Burlingame, CA, USA). Visualization of peroxidase was obtained

by incubation of the sections in a peroxidase substrate composed of

0.05% diaminobenzidine, 0.01% Nickel ammonium sulphate and

0.03% H2O2 (33%) dissolved in Tris buffer pH 7.6. The reaction was

controlled under the microscope until desired stain intensity

developed. Sections were then counterstained in thionine.

All numerical data are expressed as the mean 6 SEM; n-values

refer to the number of cells recorded.

Results

The data are from 77 current-clamp recorded MDN neurons.

Localization of the recorded neurons in the limits of the nucleus

was assessed by microscopic examination of the biocytin-labelled

neurons (Fig. 1). Mean resting potential (RP) was ±55.8 6 0.6 mV

(n = 72). As previously described in detail (Niespodziany & Poulain,

1995), regenerative LTS were produced by a depolarizing current

pulse when the neuron was held at a hyperpolarizing level ranging

from ±70 to ±90 mV, or at the termination of a hyperpolarizing

current pulse. The LTS gave rise to a single or a series of two to ®ve

Na+ action potentials.

In a ®rst series of experiments, rhythmic bursting discharges were

observed during bath application of NMDA (10±50 mM) in six

neurons among the eight tested. At the RP, NMDA produced 5±15-

mV depolarizations with tonic action potential discharges (not

illustrated). Application of continuous hyperpolarizing current to

obtain membrane potentials more negative than ±60 mV produced

rhythmic bursting discharges (Fig. 2A). The mean duration and

FIG. 1. Neurons injected with biocytin in the MDN. (A) Thionin-counterstained section cut from a frontal hypothalamic slice to show thelimits (open arrows) of the MDN, located dorsomedially to the fornix (fx),and a biocytin-®lled neuron (black arrow). (B) Biocytin-®lled MDN neuronbefore counterstaining. Scale bar, 150 mm (A), 75 mm (B).

658 P. Poulain

ã 2001 Federation of European Neuroscience Societies, European Journal of Neuroscience, 14, 657±665

frequency of the bursts were 1.2 6 0.2 s and 0.29 6 0.04 Hz

(n = 6), respectively. Intraburst discharge frequencies were highly

variable (36.3 6 8.7 Hz, n = 6). Bursts were not observed when

NMDA was applied concomitantly with 50 mM of the selective

NMDA receptor antagonist AP5 (n = 2; not illustrated).

The next experiments (69 neurons) were performed using

iontophoretic ejection of NMDA in the vicinity of the recorded

neuron, whereas all other substances were applied in the ACSF.

Preliminary trials had shown that a concentration of 50 mM of NMDA

in the iontophoretic micropipette reliably produced responses with

1±30 nA negative current, and this concentration was chosen for the

experiments. In some neurons, when the tip of the iontophoretic

micropipette was placed at the slice surface, a dramatic rise in the

membrane potential accompanied by an acceleration of ®ring leading

to depolarizing block was elicited. These neurons were not studied

further. The majority of neurons (53 of 69, 77%) responded to

iontophoretic application of NMDA with rhythmic bursting dis-

charges that were similar to those evoked during ACSF application of

the agent. Figure 2B shows a typical example of the discharges. In

this example, at the RP, a brief iontophoretic current application

produced a depolarization eliciting a tonic discharge. During

sustained iontophoretic ejection, a rhythmic bursting discharge was

generated when the membrane was manually hyperpolarized, and

bursts persisted as long as the hyperpolarization was maintained. The

rhythmicity remained constant throughout the iontophoretic applica-

tion for a given value of the membrane potential. This value ranged

from ±60 to ±78 mV (±67.4 6 0.9 mV, n = 28). Slight modi®cation

in the value of the membrane potential led to changes in burst shape.

Their duration increased as the membrane potential was less

hyperpolarized (Fig. 2C). The mean duration, frequency and

intraburst discharge frequency of the bursts were 1.47 6 0.19 s,

0.31 6 0.02 Hz and 22.6 6 3.2 Hz, respectively (n = 28).

In four additional neurons, glutamate (20 mM) was applied through

the perfusion medium and caused a membrane depolarization and an

increase in ®ring rate. Rhythmic bursting discharges appeared when

the membrane potential was hyperpolarized (Fig. 2D).

Pharmacological studies were performed on neurons in which

rhythmical bursting discharges were elicited by sustained iontopho-

teric ejections of NMDA. Perfusion of the slice with AP5 (50 mM,

n = 4) eliminated burst discharges (not illustrated). After exposure to

TTX (1 mM, n = 6), which abolished all spiking activity, neurons still

generated membrane oscillations (Fig. 3A). The mean amplitude of

the oscillations was 47 6 2.4 mV (n = 6). To examine whether

rhythmic bursting discharges depended on a sodium pump activated

by increase in intracellular Na+ concentration, ouabain (10 mM,

n = 3) and strophanthidin (10 mM, n = 3) were applied. Neither of the

substances altered the pattern of rhythmic activity (Fig. 3B and C,

Table 1). Omitting Mg2+ in the ACSF (n = 6) abruptly stopped the

rhythmic bursting discharge, which was replaced by a maintained

depolarization sustaining a tonic discharge. This effect was reversible

(Fig. 3D). The contribution of Ca2+ in the genesis of rhythmic bursts

was studied in ®ve neurons perfused with Ca2+-free ACSF (not

illustrated). This resulted in a profound modi®cation of the activity

with either a suppression of the bursts (n = 3) or an increase in burst

frequency and duration leading to a plateau and a tonic discharge

(n = 2). To determine the possible role of a transmembrane Ca2+

in¯ux through voltage-gated channels in the rhythmic bursting

discharge, Ni2+ was applied at concentrations of 40 mM (n = 3;

Fig. 4A and B) and 150 mM (n = 4; non illustrated) and nifedipine at

a concentration of 7 mM (n = 4; Fig. 4C). These blockers had no

effects on the NMDA-induced bursts (Table 1). When Ni2+ was used,

the LTS evoked by depolarizing current pulses in control conditions

were suppressed (Fig. 4B). The possible role played by a release of

Ca2+ from intracellular stores in the rhythmic bursting discharges was

examined by adding thapsigargin (2 mM, n = 4). After 40 min, the

rhythmic activity was not modi®ed (Fig. 4D, Table 1). Apamin

(2 mM, n = 6) produced a marked increase in the duration of the

bursts whereas their frequency decreased (Fig. 4E, Table 1).

Iberiotoxin (100 nM, n = 4) did not affect the pattern of rhythmic

activity (Table 1).

In a subset of neurons (9 of 69, 13%), application of NMDA by the

iontophoretic micropipette allowed the cell to ®re in repetitive LTS.

These LTS sustained bursts occurring regularly at frequencies

ranging from 0.3 to 3 Hz (1.7 6 0.2, n = 9). In Fig. 5A and B, the

FIG. 2. Rhythmic bursting discharges observed during application of NMDAthrough the bath or by iontophoresis and during application of glutamate onfour MDN neurons. (A) Bursts were observed during bath application ofNMDA (20 mM), when the membrane potential was lowered to ±63 mV(RP = ±58 mV) by a continuous negative current. A burst is shown again ata faster time scale on the right. (B) A brief iontophoretic application of±10 nA of NMDA elicited a depolarization and a tonic discharge of actionpotentials. During longer application with ±12 nA, rhythmic burstingdischarge was generated when the membrane was hyperpolarized from theRP (±52 mV) to ±72 mV (double line). (C) Continuous application of ±7nAof NMDA triggered rhythmic bursting discharges at a membrane potentialof ±67 mV (RP = ±53 mV). When the hyperpolarizing current was reducedto obtain a membrane potential of ±57 mV (double line), duration of theburst increased. In this ®gure and following ®gures, the number at the leftof the trace corresponds to the value of the iontophoretic current. (D) Bathapplication of glutamate (20 mM) depolarized the membrane (asterisk) andproduced a tonic discharge of action potentials which was converted into arhythmic bursting discharge when the membrane potential was loweredfrom ±63 mV to ±77 mV (double line).

Two types of NMDA-induced bursts in hypothalamus 659


illustrated cells ®red tonically at the RP in a single-spike mode but

exhibited a rhythmic LTS-burst ®ring in the presence of iontophor-

etically applied NMDA when the membrane was manually

hyperpolarized. Rhythmic LTS discharges persisted for all the

duration of the NMDA application. The value of the membrane

potential required ranged from ±68 to ±78 mV (±73.2 6 1.3 mV,

n = 9). As shown in Fig. 5B and C, the shape of NMDA-induced

repetitive LTS were strongly voltage-dependent. When the membrane

was less and less hyperpolarized, the width of the LTS and the

number of action potentials per burst increased whereas their

amplitude decreased. Finally, further reduction of hyperpolarization

totally abolished the LTS and gave rise to a tonic discharge (not

illustrated). NMDA-induced rhythmic LTS-burst ®ring was sup-

pressed by the application of AP5 (50 mM, n = 2; Fig. 5C). It was not

eliminated in the presence of TTX (1 mM, n = 2; not illustrated) but

totally suppressed by 40 mM of Ni2+ (n = 3; Fig. 5D). Five cells

which ®red in a rhythmic LTS pattern under NMDA were submitted

to increasing amounts of NMDA. During this procedure, the rhythmic

LTS-burst ®ring was converted into the characteristic rhythmic

bursting discharges observed for the majority of cells. Two examples

are given in Fig. 6. In the ®rst example, touching the slice surface

with the iontophoretic micropipette in the absence of ejection current

was enough to induce a rhythmic LTS-burst ®ring. Application of a

continuous iontophoretic current that increased the amount of NMDA

at the vicinity of the cell produced membrane depolarization and

tonic ®ring, and progressively the cell adopted a rhythmic bursting

activity which it could maintain for as long as the NMDA application

was continued. (Fig. 6A). In the second example, as the iontophoretic

FIG. 3. Rhythmic bursting discharges observed during continuous iontophoretic application of NMDA. (A) Responses in control ACSF and after TTX (1 mM)application. RP was ±60 mV; membrane was hyperpolarized to ±67 mV with continuous negative current. (B) In another neuron, rhythmic bursting dischargeswere still observed after perfusion of ouabain (10 mM). RP was ±52 mV; membrane was hyperpolarized to ±69 mV with continuous negative current. (C) Inyet another neuron, no modi®cation in ®ring was observed after perfusion of strophantidin (10 mM). RP was ±57 mV, membrane was hyperpolarized to±70 mV with continuous negative current. (D) Suppression of rhythmic bursting discharges by Mg2+-free ACSF in a fourth neuron´ In the upper trace, burstswere eliminated when the neuron was perfused with ACSF devoid of Mg2+ bursts reappeared following washing with normal ACSF. Samples taken at timesindicated by triangles are shown at a faster time scale on the right. The lower trace shows the moment of the transition between the rhythmic burstingdischarge in normal ACSF and the tonic discharge in the absence of Mg2+. RP was ±55 mV; membrane was hyperpolarized to ±75 mV with continuousnegative current.

660 P. Poulain


current was increased, the ®ring pattern induced by NMDA switched

from rhythmic LTS-burst ®ring to rhythmic bursting discharges

(Fig. 6B).

Discussion

The main results of the present paper are that MDN neurons are

capable of generating two distinct types of repetitive bursting activity

when they are submitted to application of NMDA. The addition of

NMDA antagonists abolished the two types of discharges, suggesting

that they are due to the speci®c activation of NMDA receptors. They

could be recorded in the same cell and the transformation of one type

of discharge to the other was obtained by modifying the amount of

NMDA.

A ®rst type of rhythmic activity, which is observed in the

majority of MDN neurons, is related to the voltage-dependent

block of the NMDA channel by Mg2+, as shown by the

experiments with Mg2+-free ACSF. Although it is the voltage

sensitivity of the NMDA channels which determines rhythmic

bursting discharges in a number of preparations, this pattern of

®ring is not common to all neurons possessing NMDA receptors.

In some systems, modulators can mask NMDA-induced bursting

(Luhmann & Prince, 1990; Sillar & Simmers, 1994; Khateb et al.,

1997; Rioult-Pedotti, 1997; MacLean et al., 1998; Schmidt et al.,

1998; Paladini et al., 1999).

In the present study, the ionic characteristics of this ®rst type of

rhythmic activity have been analysed with special attention to Ca2+,

which is, with Na+, the important carrier of NMDA-induced rhythmic

bursting in the other systems. Membrane hyperpolarization is a

prerequisite for bursting, as observed in other preparations (Hu &

Bourque, 1992; Sera®n et al., 1992; Tell & Jean, 1993). Results

obtained with TTX indicate that rhythmic bursting discharges are

driven by endogenous mechanisms and not in¯uenced by the entry of

Na+ through TTX-sensitive Na+ channels. Transmembrane Na+

currents through TTX-insensitive channels or NMDA-gated channels

are responsible ®rst for the depolarizing phase of the bursts in some

neurons (Flatman et al., 1986; Wallen & Grillner, 1987; Sera®n et al.,

1992; Rioult-Pedotti, 1997) and second for the repolarization of the

bursts in midbrain dopaminergic neurons (Johnson et al., 1992; Li

et al., 1996). In the latter, the rapid increase in intracellular Na+

concentration activates a ouabain-sensitive Na+/K+ pump ending the

burst by hyperpolarization. In the present study, the in¯uence of

sodium-free solutions on rhythmic bursts was not tested. However,

application of ouabain and strophantidin did not alter the pattern of

rhythmic bursting, demonstrating that a Na+/K+ pump is not involved

in the cessation of the burst in MDN neurons. As in other systems (Hu

& Bourque, 1992; Tell & Jean, 1993; Kim & Chandler, 1995), Ca2+

in¯ux is required for NMDA-induced rhythmic bursts in MDN

neurons because bursts are eliminated in Ca2+-free ACSF. The ways

for Ca2+ in¯ux are NMDA-gated channels themselves and voltage-

dependent Ca2+ channels. Additionally, Ca2+ release from intra-

cellular stores may contribute to the maintenance of the bursting

behaviour. There is now evidence in different neuronal systems that

Ca2+ in¯ux through high-threshold voltage-dependent Ca2+ channels

are involved in the NMDA-induced bursts recorded in physiological

Mg2+. For example, the rhythmic discharges described by Flatman

et al. (1986) in the rat sensorimotor cortex in fact consisted of pure

TABLE 1. Quantitative characteristics of the bursts before and after bath application of different substances

Substance/Parameter

Burst characteristics

Before application After application Before vs. after

Ouabain 10 mM

Duration (s) 2.1 6 0.2 2.1 6 0.1 P = 0.89Interburst frequency (Hz) 0.23 6 0.01 0.22 6 0.01 P = 0.34Amplitude (mV) 19.6 6 2.4 19 6 2.3 P = 0.55

Strophantidin 10 mM

Duration (s) 0.57 6 0.04 0.58 6 0.05 P = 0.88Interburst frequency (Hz) 0.25 6 0.03 0.26 6 0.01 P = 0.52Amplitude (mV) 24.0 6 0.6 22.5 6 1.0 P = 0.08

Nickel 150 mM


Nifedipine 7 mM


Thapsigargin 2 mM


Apamin 2 mM

Duration (s) 1.44 6 0.07 6.36 6 0.15 P < 0.0001*Interburst frequency (Hz) 0.21 6 0.01 0.031 6 0.001 P < 0.0001*Amplitude (mV) 21.68 6 0.71 22.1 6 0.8 P = 0.59

Iberiotoxin 100 nM


Means 6SEM were calculated from 10 measurements taken in a representative single neuron. Statistical signi®cance was assessed using paired Student's t-test:P < 0.05 was considered statistically signi®cant. On the basis of the selected parameters, no substances, apart from apamin, have signi®cant effects (*).



regenerative Ca2+ events. In turtle spinal mature motoneurons, they

depended on L-type channels because they were blocked by

nifedipine (Guertin & Hounsgaard, 1998). In the hippocampus, a

concomitant activation of NMDA receptors and L-channels was

required to sustain the oscillations of membrane potential (Bacci

et al., 1999). Therefore, to test the possible involvement of high-

threshold voltage-dependent Ca2+ channels in the rhythmic bursting

activity evoked by NMDA in the majority of the MDN neurons, a

concentration of Ni2+ known to block (nonspeci®cally) Ca2+ chan-

nels, and nifedipine, a speci®c L-type channel blocker, were tested. In

the presence of these antagonists, NMDA still induced rhythmic

bursting, demonstrating that entry of Ca2+ through high-threshold

voltage-dependent channels probably does not contribute to NMDA

effects. As membrane hyperpolarization, a situation that de-

inactivates T-type channels, is a prerequisite to obtain NMDA-

induced rhythmic bursting activity in MDN neurons, it is possible that

the burst requires the contribution of these channels in the early phase

of their depolarization. The observation that Ni2+, either at a low

concentration that blocks preferentially the T-type current or at a

higher concentration that suppress Ca2+ currents, did not alter

rhythmic bursting probably indicates that T-type current does not

participate in the generation of this kind of NMDA-induced bursts.

The release of Ca2+ from intracellular stores is a component of the

NMDA response (Simpson et al., 1993). A contribution of this release

to the maintenance of NMDA-induced rhythmic bursting discharges

has been suggested by Rioult-Pedotti (1997) in the lamprey.

According to this author, bursting was maintained in Ca2+-free

ACSF, but suppressed by substances known to alter the release of

Ca2+ from intracellular stores. This was not the case for MDN

neurons, because Ca2+-free ACSF abolished rhythmic bursts.

Nevertheless, Ca2+ entry, either by NMDA-gated channels or by

voltage-dependent channels, can cause the release of Ca2+ from

intracellular stores (Berridge, 1993). Furthermore, activation of

NMDA receptors can induce a thapsigargin-sensitive increase of

cytosolic Ca2+ (Gauchy et al., 2000). All these data lead to the

hypothesis that internal release of Ca2+ may activate intracellular

mechanisms capable of regulating phasically the properties of some

cellular mechanisms involved in the rhythmic activity. The observa-

tion that thapsigargin does not alter the bursts evoked by NMDA

suggests that, in MDN neurons, the release of reticular stores of Ca2+

(Thastrup et al., 1990) is not critical for the maintenance of bursting.

In accord with the scheme given by Tell & Jean (1993) and Hu &

Bourque (1992), the key factor for burst termination in MDN neurons

is probably the activation of a Ca2+-dependent K+ conductance

FIG. 4. Rhythmic bursting discharges observed during iontophoretic application of NMDA. (A) Responses in control ACSF and after Ni2+ (40 mM)application: rhythmic bursting discharges were preserved. RP was ±57 mV; membrane was hyperpolarized to ±71 mV with continuous negative current. (B)In the same cell as in A, LTS (arrow) was elicited by application of 50 ms depolarizing current pulses in control ACSF when the membrane washyperpolarized to ±71 mV (upper trace), and it was suppressed during Ni2+ (40 mM) application (lower trace). (C) In a different neuron, the upper trace is theresponse in control ACSF and the lower trace shows that rhythmic bursting activity was not modi®ed after nifedipin (7 mM) application. RP was ±52 mV,membrane was hyperpolarized to ±77 mV (double line). (D) In a third neuron, rhythmic bursting activity was still observed after 40 min incubation inthapsigargin (thaps, 2 mM). RP was ±55 mV; membrane was hyperpolarized to ±69 mV with continuous negative current. (E) In a fourth neuron, responses toNMDA in control ACSF and under apamin (2 mM) application: observe the increase in duration of the bursts and the decrease in their frequency. RP was±57 mV; membrane was hyperpolarized to ±68 mV with continuous negative current.

662 P. Poulain


consecutive to the rise of the intracellular concentration of Ca2+

during the active phase of the burst (as previously discussed, the

internal accumulation of Ca2+ is in all likelihood due to in¯ux of Ca2+

through the NMDA-gated channels). Indeed, the present study shows

that apamin increased the duration of the bursts, indicating that

repolarization of the bursts is initiated by activation of an SK-type

Ca2+-dependent K+ conductance, whereas iberiotoxin, a selective

blocker of BK-type Ca2+-dependent K+ conductance, was ineffective.

The relatively modest effect of apamin observed in present study is in

accord with previous reports (Tell & Jean, 1993; Kim & Chandler,

1995) but is in contrast with a report by Hu & Bourque (1992) where

rhythmic bursts induced by NMDA were totally blocked by apamin.

According to el Manira et al. (1994), the strength of the apamin effect

may differ from one system to another and may depend on the level

of the NMDA drive used and on the rate of bursting. Nevertheless,

the incomplete blockade observed during application of apamin

suggests that unknown mechanisms participate in the repolarization

of the bursts.

FIG. 5. LTS discharges observed during iontophoretic application of NMDA. (A) Application of NMDA elicited a sustained repetition of individual LTSwhen the membrane was hyperpolarized from the RP (±57 mV) to ±75 mV (double line). Repetitive LTS are shown at a faster time scale on the right. (B) Inanother neuron, application of NMDA elicited a sustained repetition of individual LTS when the membrane was hyperpolarized from the RP (±55.5 mV) to±68 mV (double line 1) and to ±59 mV (double line 2). Samples taken at times 1 and 2 are shown again at a faster time scale on the right. Rhythmic LTS-burst ®ring was present at the hyperpolarized levels but at the less hyperpolarized level (2) the amplitude of LTS decreased whereas the width and thenumber of triggered action potentials increased. (C) In a third neuron, rhythmic LTS-burst ®ring during continuous application of NMDA was shown atdifferent membrane potentials (upper trace: 1, ±73 mV; 2, ±63 mV; 3, ±61 mV; RP = ±53 mV). Perfusion with AP5 (50 mM) totally suppressed the LTS-burst®ring (lower trace). (D) In a fourth neuron, rhythmic LTS-burst ®ring was observed during continuous application of NMDA at a membrane potential of±78 mV (RP = ±62 mV). Firing was suppressed after Ni2+ (40 mM) application. Repetitive LTS are shown at a faster time scale on the right.



In some MDN neurons, application of NMDA led to a second type

of rhythmic activity that consisted of repetitive LTS. In control

conditions, MDN neurons never spontaneously ®re in rhythmic LTS.

Upon release of a hyperpolarizing current, they rarely ®re in a series

of repetitive LTS (Niespodziany & Poulain, 1995). The present study

demonstrates that speci®c activation of NMDA receptors induces

rhythmic LTS-burst ®ring for as long the agonist application is

continued. The involvement of LTS in this striking pattern of ®ring is

obvious because Ni2+, by suppressing the T-type current, totally

blocks the bursts. The possibility that this blocking effect is due

instead to a modi®cation of the NMDA-gated channel (Mayer &

Westbrook, 1987) is small, because Ni2+ was without effect on the

other type of NMDA-induced bursting observed in MDN neurons.

Rhythmic LTS-burst ®ring due to NMDA has only been observed

twice, in thalamocortical neurons (Leresche et al., 1991), and in

nucleus basalis cholinergic neurons (Khateb et al., 1995), and

analysed in the latter study: in contrast to the Mg2+ requirement in the

other type of NMDA-induced rhythmic bursts, Mg2+ was not found to

be necessary for the occurrence of repetitive LTS. It has been

reported that low doses of NMDA can facilitate T-type channels (Bon

et al., 1998) and can enhance and prolong LTS (Khateb et al., 1995;

Bon et al., 1998), but how NMDA may promote the occurrence of a

repetitive discharge of LTS is unknown. In the present study, raising

the amount of NMDA reversibly transformed the rhythmic LTS-burst

®ring into the rhythmic bursting discharges observed in most of the

MDN cells and in the majority of the other systems. Such a switch

from one kind of burst ®ring to another has not previously been

described. Thus, the present study demonstrates for the ®rst time that

the two distinct types of bursting can coexist in the same neuron. It

also gives evidence that these two types of bursting are independent.

Ineffectiveness of Ni2+ in modifying the rhythmic bursting dis-

charges, whereas LTS were suppressed, clearly indicates that the T-

type current (IT) is not involved in this type of bursting. In the present

experiments, only 13% of the neurons were able to display rhythmic

LTS-burst ®ring. This is in marked contrast to the great proportion of

neurons where rhythmic bursting discharges can be triggered. A ®rst

explanation may be that only a subpopulation of MDN LTS neurons

possesses the conductances required to ®re in repetitive LTS under

NMDA. An alternative explanation may be that the level of NMDA

for switching from a mode to another is very critical and that, in most

of the cases, the threshold for triggering the rhythmic bursting

discharges that is observed in the majority of the neurons is

overstepped even with the lowest currents used to eject NMDA.

As glutamate was able to trigger rhythmical bursting discharges

in the present study, it is likely that NMDA receptor activation by

glutamatergic afferents can favour rhythmic bursting in vivo. In

the present experiments, bursting required the injection of

hyperpolarizing currents. This implies that activation of a

concurrent inhibitory in¯uence is necessary for natural bursting

in MDN neurons, perhaps by inhibitory synaptic inputs coming

from local gamma-aminobutyric acid interneurons (Boudaba et al.,

1996).

The role of bursting in the physiological activity of the MDN

neurons is necessary speculative. These neurons synthesize enkepha-

lins and project to the lateral septum (Poulain et al., 1984), where

enkephalins exert an inhibitory action (Carette & Poulain, 1982). The

information carried by the MDN neurons depends on their temporal

pattern of neuronal activity. Thus, bursting could generate conditions

for increasing the synthesis of enkephalin and/or for increasing the

synaptic ef®cacy during release of enkephalin. The present study

suggests that the strength of the glutamatergic drive may determine

one or another type of bursting. This process may serve to subtly

modulate the control exerted by enkephalins in the lateral septum,

depending on the activity of the glutamatergic afferents to the MDN

neurons. Projections from the lateral septum directly reach the

hypothalamus (Varoqueaux & Poulain, 1999) and are involved in the

multiple functions of the hypothalamus modulated by the hippocam-

pus (review in Jakab & Leranth, 1995). Thus, bursting activity

evoked by NMDA-receptor activation in the MDN may be of

importance in the control by enkephalins of the hippocampal signal

FIG. 6. Switch from the rhythmic LTS-burst®ring to the rhythmic bursting activity duringiontophoretic application of NMDA. (A) Onthe left, diffusion of NMDA from theiontophoretic micropipette generated arhythmic LTS-burst ®ring when the membranewas hyperpolarized to ±78 mV with continuousnegative current (RP = ±62 mV). On the right,a rhythmic bursting activity developed in thesame neuron at the same membrane potentialwhen an iontophoretic current (±14 nA) wasapplied to eject NMDA from the micropipette.(B) From another neuron, a rhythmic LTS-burst ®ring was triggered by iontophoreticallyapplied NMDA when the membrane washyperpolarized to ±77 mV with continuousnegative current (RP = ±56 mV). Increasingthe iontophoretic current from ±3 nA to ±9 nA(arrow) brought about a switch from therhythmic LTS-burst ®ring to the rhythmicbursting activity: lower traces are samples ofthe two types of ®ring at a faster time-scale.

664 P. Poulain


¯ow that relays in the lateral septum to in¯uence hypothalamic

functions.

Abbreviations

ACSF, arti®cial cerebrospinal ¯uid; AP5, DL-2-amino-5-phosphonopentanoicacid; LTS, low-threshold Ca2+ spike; MDN, magnocellular dorsal nucleus;NMDA, N-methyl-D-aspartate; RP, resting potential; TTX, tetrodotoxin.

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two distinct types of repetitive bursting activity mediated by nmda in hypothalamic neurons in vitro

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