Swelling-activated calcium signalling in cultured mouseprimary sensory neurons

FeÂlix Viana, Elvira de la PenÄa, Brian Pecson, Robert F. Schmidt and Carlos BelmonteInstituto de Neurociencias, Universidad Miguel HernaÂndez-CSIC, Apartado 18, San Juan de Alicante 03550, Spain

Keywords: cell volume, chloride channels, intracellular calcium stores, mechanotransduction, trigeminal neuron

Abstract

The effects of hypo-osmotic membrane stretch on intracellular calcium concentration ([Ca2+]i), cell volume and cellular excitabilitywere investigated in cultured mouse primary sensory trigeminal neurons. Hypotonic solutions (15±45%) led to rapid cell swelling

in all neurons. Swelling was accompanied by dose-dependent elevations in [Ca2+]i in a large fraction of neurons. Responses

could be classi®ed into three categories. (i) In 57% of the neurons [Ca2+]i responses had a slow rise time and were generally ofsmall amplitude. (ii) In 21% of the neurons, responses had a faster rise and were larger in amplitude. (iii) The remaining cells

(22%) did not show [Ca2+]i responses to hypo-osmotic stretch. Slow and fast [Ca2+]i changes were observed in trigeminal

neurons of different sizes with variable responses to capsaicin (0.5 mM). The swelling-induced [Ca2+]i responses were not

abolished after depletion of intracellular Ca2+ stores with cyclopiazonic acid or preincubation in thapsigargin, but were suppressedin the absence of external Ca2+. They were strongly attenuated by extracellular nickel and gadolinium. Hypotonic stimulation led

to a decrease in input resistance and to membrane potential depolarization. Under voltage-clamp, the [Ca2+]i elevation produced

by hypotonic stimulation was accompanied by the development of an inward current and a conductance increase. The timecourse and amplitude of the [Ca2+]i response to hypo-osmotic stimulation showed a close correlation with electrophysiological

properties of the neurons. Fast [Ca2+]i responses were characteristic of trigeminal neurons with short duration action potentials

and marked inward recti®cation. These ®ndings suggest that hypo-osmotic stimulation activates several Ca2+-in¯ux pathways,including Gd3+-sensitive stretch-activated ion channels, in a large fraction of trigeminal ganglion neurons. Opening of voltage-

gated Ca2+ channels also contributes to the response. The pattern and rate of Ca2+ in¯ux may be correlated with functional

subtypes of sensory neurons.

Introduction

Peripheral endings of mammalian primary sensory neurons respond

to a variety of stimuli. Based upon the class of energy (mechanical,

thermal or chemical) by which the receptor endings are preferentially

activated, several functional types of sensory neurons have been

identi®ed (Campbell & Meyer, 1996). Sensory neurons can be further

subdivided into high- and low-threshold units according to the

intensity of the stimulating force to which they respond (Burgess &

Perl, 1973).

Knowledge of the cellular and molecular mechanisms involved in

the transduction and encoding of stimuli by mammalian primary

somatosensory neurons is important for the understanding of the

sensory discriminative capacities of the nervous system. A major

obstacle for progress lies in the small size of peripheral nerve

endings. A membrane receptor associated with responses of

nociceptive neurons to capsaicin, protons and heat has been identi®ed

and cloned (Caterina et al., 1997, 2000; Tominaga et al., 1998). Other

ion channels selectively expressed in primary sensory neurons and

involved in acid sensing have also been characterized (Bevan &

Yeats, 1991; Waldmann & Lazdunski, 1998). However, the

transduction processes in peripheral sensory neurons exhibiting low

and high mechanical thresholds, and responsible for the senses of

touch and mechanical pain, remain poorly de®ned. Several types of

stretch-activated channels have been described (reviewed by Garcia-

AnÄoveros & Corey, 1997), with diverse biophysical and pharmaco-

logical properties. Recently, a mechanosensory transduction channel

has been cloned in Drosophila and found to be expressed in

invertebrate mechanosensory neurons selectively (Walker et al.

2000). In aortic baroreceptor neurons, membrane stretch led to

elevations in intracellular Ca2+ via in¯ux through Gd3+-sensitive

channels (Cunningham et al., 1995; Sullivan et al., 1997). Another

study suggested a role for epithelial sodium channels (ENaC)

channels in the mechanical response of baroreceptors (Drummond

et al., 1998). Also recently, a mechanosensitive chloride channel was

identi®ed at the terminal endings of cultured rat dorsal root ganglion

(DRG) neurons (Imai et al., 2000). In a study by Gotoh & Takahashi

(1999), [Ca2+]i rises were evoked in a fraction of DRG neurons by

mechanical deformation of the cell surface. These [Ca2+]i changes

presumably re¯ect the opening of stretch-activated channels located

in the cell soma (McCarter et al., 1999). Nevertheless, responses

evoked by controlled stretch in the various functional types of

primary sensory neurons have not been measured.

Rapid changes in cell volume can be evoked by changes in

extracellular osmotic pressure. In many nonexcitable cells hypo-

osmotic cell swelling is associated with elevations in [Ca2+]i (Oike

et al., 1994; Chen et al., 1996; Altamirano et al., 1998). A similar

increase in [Ca2+]i has been documented in other mechanosensory

transducers, like sensory hair cells (Harada et al., 1994) and putative

Correspondence: Dr FeÂlix Viana, as above.Email: felix.viana@umh.es

Received 10 August 2000, revised 24 November 2000, accepted 8 December2000

European Journal of Neuroscience, Vol. 13, pp. 722±734, 2001 ã Federation of European Neuroscience Societies

baroreceptors (Cunningham et al., 1995), and the favoured

mechanism of Ca2+ entry during swelling is the opening of stretch-

activated ion channels (Christensen, 1982; reviewed by McCarty &

O'Neil, 1992).

Trigeminal ganglion (TG) neurons are primary sensory neurons

that innervate deep and super®cial tissues of the head and face. In the

present study we used fura-2-based micro¯uorimetric calcium

imaging in combination with whole-cell patch-clamp recordings to

investigate the mechanisms responsible for [Ca2+]i elevations in

different subpopulations of primary sensory neurons of the TG

following hypo-osmotic stretch. We also investigated the effects of

cell swelling upon neuronal excitability. Our results reveal a

functional specialization of TG neurons in culture and support the

participation of multiple mechanisms, including anion and cation

conductances, in the transduction of hypo-osmotic stimuli. A

preliminary report of this work has been published in abstract form

(de la PenÄa et al., 1999).

Materials and methods

Culturing of trigeminal primary sensory neurons

Methods for culturing trigeminal primary sensory neurons were

similar to those used previously in our laboratory (Garcia-Hirschfeld

et al., 1995). In brief, male and female newborn mice (from P2 to P5)

were anaesthetized with ether and their trigeminal ganglia (TG)

excised. The TG were washed in phosphate buffered saline (PBS)

solution (Sigma) and collected in cold L15 solution (Sigma) with

gentamycin (50 mg/mL). Ganglia were cut in small pieces and

incubated for 45 min in warm DMEM (37 °C, gassed with 95% O2/

5% CO2) containing 0.25% collagenase (380 units/mg, type IA,

Sigma). They were gently triturated with a ®re-polished glass pipette

and the resulting solution was centrifuged at 1000 r.p.m. for 8 min.

The pellet obtained was resuspended in culture medium (317 mOsm/

kg) with the following composition: 45% Dulbecco's modi®ed

Eagle's medium (DMEM), 45% F-12, 10% fetal calf serum (GIBCO),

supplemented with 4 mM L-glutamine (GIBCO), 100 mg/mL peni-

cillin/streptomycin, 20 mM glucose, nerve growth factor (NGF,

mouse: 7S, 100 ng/mL, Sigma). Cells were plated (80 mL) on

round glass coverslips (12 mm) in small plastic Petri dishes (35 mm)

and kept at 37 °C gassed with 95% O2/5% CO2. Coverslips had

already been treated with poly L-lysine (0.01%, Sigma) and dried.

Plated TG neurons appear as round-to-ovoid cells with a variety of

diameters. Their diameter varied between 10 and 30 mm. Their

diameters showed a Gaussian distribution, with a maximum between

18 and 20 mm.

Solutions and hypotonic stimuli

Solutions of different osmolarities were employed. The osmolarity of

each solution was measured using a cryoscopic osmometer (Gonotec,

Berlin, Germany). Unless speci®cally noted in the text, to maintain a

constant ionic composition only the concentration of D-mannitol was

adjusted. The compositions of the different solutions used are listed in

Table 1. In most experiments, a 30% hypo-osmotic solution was

tested. This is at a degree of tonicity several times lower than the

value reported to suppress the release vasopressin in humans (Moses

& Miller, 1974).

Cells were continuously perfused (2±4 mL/min) in the recording

chamber with isotonic solution. The perfusion could be switched to

one of several test solutions via a multibarelled stopcock. Exchange

of solutions was completed in » 20±30 s. In experiments testing the

effects of Gd3+, bicarbonate was substituted with 10 mM HEPES

(Caldwell et al., 1998). Some solutions contained tetrodotoxin citrate

(TTX) (Tocris, Bristol, UK) at a concentration of 1 mM.

Electrophysiological recordings

Patch-pipettes fabricated of borosilicate glass (Clark, UK), with a

resistance of 3±8 MW when ®lled with the standard pipette solution,

were employed. Membrane currents were recorded with a RK300

patch-clamp ampli®er (Biologic, Meylan, France) in voltage- and

TABLE 1. Composition of the different extracellular solutions used

Osmolality(mOsm/kg)

NaCl(mM)

KCl(mM)

KH2PO4

(mM)MgSO4

(mM)MgCl2(mM)

CaCl2(mM)

NaHCO3

(mM)Glucose(mM)

D-mannitol(mM)

Isotonic 314 74 5 1.2 1.3 0 2.4 26 10 100Hypotonic 30% 209 74 5 1.2 1.3 0 2.4 26 10 0Hypertonic 30% 421 74 5 1.2 1.3 0 2.4 26 10 200Isotonic-free Ca2+ 314 76 5 1.2 1.3 2.4 0 26 10 100Hypotonic 30% (Ca2+-free) 207 76 5 1.2 1.3 2.4 0 26 10 0Isotonic-Hepes 310 74 5 0 0 1.3 2.4 0 10 116Hypotonic 30%-Hepes 200 74 5 0 0 1.3 2.4 0 10 16Isotonic * 320 50 5 1.2 1.3 0 2.4 26 10 148Hypotonic 15% * 263 50 5 1.2 1.3 0 2.4 26 10 100Hypotonic 30% * 216 50 5 1.2 1.3 0 2.4 26 10 48Hypotonic 45% * 166 50 5 1.2 1.3 0 2.4 26 10 0Hypertonic 30%² 401 174 5 1.2 0 3.7 0 26 10 0Isotonic² 308 124 5 1.2 0 3.7 0 26 10 0Hypotonic 15%² 263 99 5 1.2 0 3.7 0 26 10 0Hypotonic 30%² 217 74 5 1.2 0 3.7 0 26 10 0Hypertonic 30%³ 401 174 5 1.2 0 1.3 2.4 26 10 0Isotonic³ 308 124 5 1.2 0 1.3 2.4 26 10 0Hypotonic 15%³ 263 99 5 1.2 0 1.3 2.4 26 10 0Hypotonic 30%³ 217 74 5 1.2 0 1.3 2.4 26 10 0

The osmolality of the solutions represents the average of at least three measurements. The solutions of the ®rst ®ve rows were used for most experiments in thispaper. The bicarbonate-free, Hepes-buffered solutions contained 10 mM HEPES and were only used in experiments testing the blocking effect of gadolinium.*Solutions (lower NaCl/higher D-mannitol) used only in dose±response experiments (Fig. 3A and B). ²Solutions used in experiments examining ¯uorescencechanges during hypotonic stimulation responses (Fig. 2A and B). ³Solutions used in experiments examining simultaneous volume and Ca2+ changes duringhypotonic stimulation responses (Fig. 2C).

Transduction in primary sensory neurons 723

ã 2001 Federation of European Neuroscience Societies, European Journal of Neuroscience, 13, 722±734

current-clamp modes. Data acquisition was performed using

pCLAMP 6-8 software (Axon Instruments).

The standard pipette solution contained (in mM): 140 KCl, 10

NaCl, 4 Mg-ATP, 0.4 Na-GTP, 0.2 fura-2 pentapotassium salt and 10

HEPES (300 mOsm/kg, pH 7.3 adjusted with KOH). In some

experiments, 100 KCl was substituted with K-aspartate.

Cells with a resting membrane potential other than ±60 mV were

hyperpolarized or depolarized to bring the membrane potential near

±60 mV. Action potential threshold was estimated during injection of

a series of 500 ms depolarizing current pulses of increasing

amplitude (current steps, 10±25 pA). The duration of the action

potential was measured 20% above the voltage threshold, de®ned

by the marked change in the time derivative of the voltage (Vm)

response. The degree of sag or inward recti®cation was quanti®ed

as a recti®cation index (RI). RI was de®ned as [Vm peak ±

Vm steady-state]/Vm peak during hyperpolarizing voltage responses that

reached a Vm peak-value » ±120 mV. During voltage-clamp experi-

ments, membrane conductance was monitored with hyperpolarizing

pulses (10 mV, 100 ms, 1 pulse/5 s) from a holding potential of ±

60 mV.

Fluorometric Ca2+ measurements

Intracellular Ca2+ measurements ([Ca2+]i) were performed in single

cells loaded with the acetoxymethylester form of fura-2 (fura2-AM,

Molecular Probes, Eugene, USA). Neurons were incubated for

30 min in oxygenated solution (95% O2 + 5% CO2) containing

fura2-AM (5±10 mM with 0.02% pluronic acid) at 37 °C. Thereafter,

neurons were gently washed with oxygenated saline and kept at room

temperature until use. Coverslip pieces were placed in a custom-

made, temperature-controlled microchamber (0.5 mL volume) at a

temperature of 32 6 2 °C and continuously perfused with oxygen-

ated solutions. Cells were viewed through an Olympus 3 60 water

immersion objective (NA = 0.90) mounted on an upright microscope

(Axioskop FS, ZEISS).

In those experiments combining [Ca2+]i measurements with whole-

cell recordings, fura-2 pentapotassium salt (Molecular Probes) was

FIG. 1. Selective activation and different time courses in the [Ca2+]i response to hypotonic stimulation. (A±D) Specimen photographs of a culture withtrigeminal primary sensory neurons (N1, N2, N3 and N4), ®broblasts and glial cells, under control (isotonic) conditions (A), during exposure to 30%hypotonic solution for 80 s (B) and 280 s (C), and after a wash-out period (D). (E) The time course of the [Ca2+]i changes during the 30% hypotonicstimulation. Note the very different response time-courses and amplitudes in the fast-type (N1) and slow-type (N2) TG neurons, and the absence of responsein the neuron marked N3. The cell marked N4 is also a neuron that lacked a clear response. (F) Histogram with the distribution of peak [Ca2+]i responses tothe 30% hypotonic solution in a sample of n = 103 neurons taken from 44 trigeminal ganglion cultures. Note that the abscissa is discontinuous and the barson the right appear much thinner. The inset in F shows the percentages of Fast-type (Fast), Slow-type (Slow) and unresponsive neurons (NR) in the samesample. (G) Scattered plot of the peak [Ca2+]i responses in 17 TG neurons tested twice with an identical stimulus (30% hypotonic for 5 min). The diagonalline represents the identity line.

724 F. Viana et al.

ã 2001 Federation of European Neuroscience Societies, European Journal of Neuroscience, 13, 722±734

also added to the pipette solution at a concentration of 200 mM and

loaded into the cell via the recording patch pipette.

Fluorometric measurements were performed using a digital

imaging system, with a cooled CCD camera (Sensys, Photometrics)

and an Image Lightning 2000 video imaging board (Axon

Instruments, CA, USA). The acquisition and on-line display of

images was performed with AIW software (Axon Instruments).

Digital ¯uorescence images of cultured neurons were obtained using

paired exposures to excitation wavelengths of 357 nm (isosbestic for

fura-2) and 380 nm. The excitation light source was a 50-W mercury

lamp. Switching to both excitation wavelenghts was obtained with a

computer controlled ®lter wheel (Lambda 10-2; Sutter Instruments,

CA, USA). Fluorescence emission at wavelengths longer than

510 nm were monitored. Images were acquired and stored at

0.2 Hz.

The background ¯uorescence was measured in a region of the

culture dish devoid of cells and subtracted on-line from the cellular

¯uorescence signals. The apparent concentration of free intracellular

calcium [Ca2+]i was calculated from the ratio (R) of the ¯uorescence

emission at the two excitation wavelengths, using the formula [Ca2+]i

= Keff*[(R ± Rmin)/(Rmax ± R)]. The parameters Rmin (¯uorescence

ratio at zero [Ca2+]i) and Rmax (limiting ¯uorescence ratio at high

[Ca2+]i levels) were obtained in vitro with solutions of a Ca2+

calibration kit (Molecular Probes). The effective binding constant

(Keff) was calculated from actual calibration values according to the

relationship Keff = 225*[(Rmax ± R)/(R ± Rmin), using a solution with

225 nM free Ca2+ and deriving R from the ratio corresponding

¯uorescence (Neher & Augustine, 1992).

Combined measurement of cell volume and intracellular Ca2+

Changes in cell water volume during hypotonic stimulation were

estimated from the change in intracellular concentration of fura-2,

recorded at the Ca2+-insensitive wavelength (isosbestic, 357 nm). The

change in fura-2 concentration results in a change in ¯uorescence

intensity that is proportional to cell-volume changes (see Fig. 2B).

Combined with recordings of the Ca2+-sensitive wavelength

(380 nm), it was possible to monitor changes in cell water volume

and [Ca2+]i in single cells simultaneously (Muallem et al., 1992;

Altamirano et al., 1998). Similar techniques to estimate changes in

cell volume have been used by Aitken et al. (1998) in dissociated

CA1 hippocampal neurons.

Because we were primarily interested in the time course of cell-

volume changes with respect to [Ca2+]i changes, and not in the

absolute changes in cell volume, we neglected ¯uorescence back-

ground due to intrinsic cell ¯uorescence (negligible in these

experiments) and osmotically insensitive component of the ¯uores-

cent dye (this component averaged 33% in our experiments, see

legend of Fig. 2B). Under these conditions, normalized cell water

volume changes (Vx/Viso) are inversely proportional to changes in

relative ¯uorescence (Fx/Fiso) resulting from excitation of fura-2 at

357 nm. Fiso represents the average ¯uorescence of a small region of

the cell in control isotonic solution having an osmotic pressure piso

FIG. 2. Combined measurement of cell volume and intracellular Ca2+. (A) Time course of ¯uorescence change in a TG neuron, AM-loaded with fura-2, andexcited alternatively at 380 nm (upper trace) and 357 nm (middle trace) during consecutive 15% and 30% hypotonic stimulations. The entire experiment wasperformed with solutions lacking external Ca2+ (see Table 1). Both traces have been linearly corrected for a time-dependent ¯uorescent loss of 2%/min.(B) Change in fura-2 ¯uorescence (357 nm excitation) as a function of external osmolarity. The cellular ¯uorescence (Fx) in solutions of different osmolarity(px) has been expressed as a percentage of the ¯uorescence measured in isotonic solution (Fiso, piso = 308 mOsm/kg). The points have been ®tted with alinear relationship (r = 0.99). Each point represents the mean value from four to 13 cells. All experiments were performed in Ca2+-free external solutions andeach cell was subject to a single anisosmotic challenge. The dotted line represents the expected relationship if all the intracellular ¯uorescence was sensitiveto osmotic changes. Note that the ratio of these two slopes represents the percentage of osmotically active ¯uorescence, which was 67%. (C) Time-course ofchange in volume (continuous line) and intracellular calcium (®lled circles) during a 5-min 30% hypotonic stimulation. Examples of three different TGneurons with fast (left), slow (centre) and no [Ca2+]i responses (right) to the osmotic stimulus. The time calibration applies to all records. The D Ca2+

calibration on the right also applies to the record in centre.

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ã 2001 Federation of European Neuroscience Societies, European Journal of Neuroscience, 13, 722±734

(see Table 1) and Fx represents the average ¯uorescence of the same

region of the cell in hypotonic or hypertonic solutions, with osmotic

pressure px. Vx and Viso are the corresponding cell water volumes. In

these series of experiments, the hypotonic and hypertonic solutions

were obtained by adding or removing NaCl from an isotonic solution

with 124 mM NaCl (see Table 1). It was not possible to use mannitol

replacement because it produced an artifactual drop in absolute

¯uorescence (ratio ¯uorescence was unaffected).

The drift in absolute ¯uorescence signal resulting from dye leakage

and photobleaching was determined for each experiment and

corrected by ®tting a linear regression to the baseline (5 min) and

multiplying the slope of this regression line by the time at which each

data point was sampled. To minimize photobleaching, the sampling

rate was reduced to 0.1 Hz, and excitation light intensity was

attenuated with neutral density ®lters. The loss of ¯uorescence

averaged 3 6 0.3% per minute of recording (n = 24).

Data are reported as means 6 SEM. Statistical signi®cance

(P < 0.05) was assessed by the Student's t-test.

Results

Types of [Ca2+]i responses to hypotonic stimulation

One to six neurons in a coverslip piece were selected for ¯uorimetric

Ca2+ measurements. For a period of 2 min, control [Ca2+]i-values were

obtained in isotonic solution (see Table 1). Thereafter, the perfusion

was switched to a 30% hypotonic solution for 5 min. The specimen

records in Fig. 1 illustrate the responses of four trigeminal neurons

(labelled N1, N2, N3 and N4) under isotonic conditions (A) and at

different times after switching to the hypotonic solution (B±C). As

shown in B, 80 s after beginning of the stimulation the neuron N1 has

increased its originally quite low [Ca2+]i markedly; N2 increased its

[Ca2+]i modestly, whereas neurons labelled N3 and N4 showed no

change in their [Ca2+]i. Some 200 s later (C) the [Ca2+]i level of neuron

N1 had declined to control level, while the [Ca2+]i level in neuron N2

remained elevated, and [Ca2+]i in N3 and N4 had remained nearly

unchanged. All neurons returned to baseline shortly after application

of isotonic solution (D). The full time course of the [Ca2+]i response in

three of the neurons (N1, N2 and N3) is illustrated in Fig. 1E.

Altogether, 103 TG neurons were exposed to the 30% hypotonic

solution. The histogram in Fig. 1F shows the distribution of peak

response amplitudes in the sample. The time courses and amplitudes of

[Ca2+]i responses to cell swelling allowed the distinction between fast-

responding (21%), slow-responding (57%) and nonresponding (22%)

neurons. Fast responses were characterized by a fast rise time to an

initial peak (mean time to peak of 34 6 3 s), followed by a rapid

decline to a plateau of variable amplitude during the remainder of the

5 min exposure to the hypotonic solution (see Fig. 6). Fast responses

were generally of large amplitude with a mean of 201 6 59 nM

(n = 22). This time course was in marked contrast with responses

recorded in slow-type neurons. In these TG neurons, [Ca2+]i responses

to the hypotonic stimulus took a considerable time to reach the peak

(mean time to peak = 172 6 9 s), and they had smaller amplitudes

(mean = 54 6 6 nM; n = 59; see Fig. 1E, N2, and Fig. 6).

In 17 neurons, a second 5-min application of a 30% hypotonic

solution was repeated after a 10±15-min interval in isotonic solution.

As shown in Fig. 1G, the response was quite reproducible in those

cells (n = 14) giving a small initial response (< 70 nM). In this case

the mean ratio of the second response amplitude to the ®rst was

FIG. 3. Swelling induced [Ca2+]i signals aredose-dependent. (A) [Ca2+]i responses in a TGneuron of the fast type to application ofsolutions of increasing hypotonicity (15, 30and 45%, respectively). Each application lastedfor 5 min. (B) Peak increase in [Ca2+]i

(average 6 SEM) obtained in trigeminalneurons when applying the protocol displayedin A. Only cells in which the three levels ofhypotonicity were tested were included in the®gure (n = 22). The data represent the meanincrease in [Ca2+]i for all cells (responding andnonresponding). The mean value for 130%tonicity (n = 6) was obtained with the protocoldisplayed in Panel C (the error bar is smallerthan the size of the ®lled circle). (C) [Ca2+]i

responses in another fast-type neuron,illustrating the absence of any change in[Ca2+]i when a 30% hypertonic solution wasapplied. (D) Peak changes in [Ca2+]i in sixneurons when applying 30% hypotonicity for5 min (black column), and the absence of anychange in [Ca2+]i during a comparableapplication of 30% hypertonicity to the samesample of neurons (grey column).

TABLE 2. Volume response in three groups of TG neurons

Ca2+ delay(s)

D Ca2+

(nM)Swelling(%)

RVD(%)

Fast Ca2+ response ±9 6 3* 285 6 87* 16 6 1² 50 6 14²(n = 6)

Slow Ca2+ response 59 6 8 74 6 12 20 6 1 52 6 7(n = 16)

No Ca2+ response NA 7 6 1 19 6 4 32 6 12(n = 5)

Neurons were grouped according to the time delay between the swellingresponse and the [Ca2+]i response to a 5-min duration 30% hypotonic stimulus.The delay was measured as the time interval between 50% of the peak volumeand 50% of the peak [Ca2+]i response. Negative values imply that the peak[Ca2+]i response preceded the peak volume response. D Ca2+ represents thedifference between resting [Ca2+]i and peak [Ca2+]i during hypotonic swelling.Regulatory volume decrease (RVD) was measured as percentage of volumerecovery with respect to peak volume increase. Statistical differences wereevaluated with a t-test between pairs of groups. The symbols (*and ²)indicatethat differences between all groups in the column were highly signi®cant(*P < 0.001) or nonsigni®cant (²P > 0.05), respectively.

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0.95 6 0.11. In contrast, the second response showed a strong degree

of adaptation in those cells with a large initial [Ca2+]i response

(> 100 nM). In these neurons (n = 3), the fast type response

converted into a slow type pattern upon a second exposure to the

hypotonic solution, and the ratio of the two responses dropped to

0.19 6 0.04.

In 17 additional neurons, the 30% hypotonic stimulation was

performed in the presence of 1 mM TTX. In 71% of those neurons

(12/17) [Ca2+]i responses were still observed (not shown), with an

average [Ca2+]i elevation of 111 6 20 nM above baseline. This result

suggested that ®ring of TTX-sensitive action potentials were not

essential for triggering the calcium responses observed.

Temporal relationship between cell volume and [Ca2+]ichanges

It has been shown previously that changes in fura-2 ¯uorescent

intensity during hypotonic stimulation can be used to estimate cell-

volume changes in osteosarcoma (Muallem et al., 1992) and

neuroblastoma cells (Altamirano et al., 1998). We validated the use

of this technique in neuronal cells. Figure 2A shows the change in

fura-2 ¯uorescence intensity during excitation at two wavelenghts

(357 and 380 nm) in a TG neuron, during consecutive applications of

15% and 30% hypotonic steps. This experiment was performed in

zero external Ca2+, to prevent changes in [Ca2+]i that would affect the

¯uorescence monitored at nonisosbestic wavelenghts (e.g. 380 nm).

The lack of change in [Ca2+]i is shown in the lower trace of Fig. 2A.

The 15% hypotonic stimulation produced a decrease in ¯uorescence

of » 10% that returned to baseline upon termination of the stimulus.

The 30% hypotonic stimulation produced a larger decrease in

¯uorescence. In addition, the level of ¯uorescence tended to sag back

towards the initial value and a marked overshoot in ¯uorescence

occurred during the return to the isotonic solution. This sag and

overshoot re¯ect the process of regulatory volume decrease (RVD); a

return from the peak swollen volume back to the initial volume

measured under isosmotic conditions, and a further shrinkage of the

cell. The process of RVD has been described in many cells (reviewed

by Lang et al., 1998). The degree of RVD was quite variable in TG

neurons, especially in the presence of external Ca2+ (see Fig. 2C).

The plot in Fig. 2B represents the average change in ¯uorescence,

measured at 357 nm, as a function of external tonicity. The

relationship was linear for a range of solutions 30% above and

below the normal isotonic solution, validating its use as an index of

relative cell volume.

We evaluated the time course of [Ca2+]i change with respect to the

volume change during a 5-min 30% hypo-osmotic stimulation, in 27

TG neurons. A summary of results is presented in Table 2. Figure 2C

shows three typical examples of changes in cell volume (continuous

line) superimposed on the corresponding [Ca2+]i changes (®lled

circles). In 22% of the neurons, the [Ca2+]i elevation occurred

simultaneously with the change in volume, the [Ca2+]i actually

peaking before the maximal volume was reached (Fig. 2C, left). In

this group of neurons, the average [Ca2+]i elevation was larger than in

the other groups (P < 0.001, Table 2). More frequently (59% of the

neurons), the change in [Ca2+]i started with a clear delay with respect

to the volume change and had a slower time course (Fig. 2C, centre).

In these cells, the [Ca2+]i response outlasted the volume recovery

upon return to isotonic conditions. Furthermore, the average [Ca2+]i

elevation was smaller in this group compared to the fast group

(Table 2). Finally, a third group of cells did not show any changes in

[Ca2+]i despite normal changes in cell volume (Fig. 2C, right). The

relative volume change and the percentage of RVD were not

statistically signi®cant among the three groups of TG neurons,

suggesting that the time course of volume change was not the primary

determinant of the [Ca2+]i response.

Responses to various degrees of hypotonicity and tohypertonicity

As illustrated in Fig. 3, [Ca2+]i changed in response to the application

of external solutions of decreasing tonicity (Fig. 3A). In this example,

a reduction in the tonicity of the external solution by 15% did not

increase the [Ca2+]i, while reductions of 30% and 45% produced

larger responses. This type of analysis was performed in a

subpopulation of 22 neurons that were tested with three levels of

hypotonicity. A summary of their dose-dependence is shown in

Fig. 3B. Furthermore, the percentage of responding cells increased

with the intensity of the stimulus. Thus, with 15% hypotonic solution

only 14% (3/22) gave a signi®cant increase in [Ca2+]i. With 30%

FIG. 4. Swelling induced [Ca2+]i signalsrequire extracellular Ca2+. (A) [Ca2+]i

responses recorded simultaneously in twocultured trigeminal neurons. The standardisotonic solution (2.4 mM Ca2+) was ®rstreplaced with one lacking external Ca2+ and100 s later by one which in addition had itstonicity reduced to 70%. After 5 min in Ca2+-free hypotonic solution, external Ca2+ wasreturned to 2.4 mM. (B) Mean [Ca2+]i levels in13 TG neurons during recordings undervarious external solutions speci®ed at thebottom of the diagram (same protocol as in A).(C) Effect of 20 mM CPA on the [Ca2+]i levelin a trigeminal neuron. The application of CPAdid not prevent the response to a subsequentapplication of 30% hypotonic solution. (D)Mean [Ca2+]i levels in seven trigeminalneurons during recordings under variousexternal solutions speci®ed at the bottom ofthe diagram (same protocol as in C). Data inisotonic-CPA (hatched column) were obtainedjust before the start of the hypotonicapplication.

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hypotonicity this percentage increased to 41% (9/22) and with 45%

hypotonicity nearly all neurons 91% (20/22) responded. In contrast, a

30% hypertonic solution (for its composition see Table 1) did not

have any effect upon [Ca2+]i (Fig. 3C and D).

Dependence of hypotonicity-induced increases in [Ca2+]i onextracellular Ca2+ in¯ux

The hypotonicity-induced increases in [Ca2+]i could be due to in¯ux

of Ca2+-ions from the external medium and/or the release of such ions

from intracellular stores. In the latter case this release may or may not

require the in¯ux of external Ca2+-ions to trigger the intracellular

release. These various mechanisms have been shown to operate in

different cell types (Oike et al., 1994; Cunningham et al., 1995; Chen

et al., 1996; Senok & Baumann, 1997).

As illustrated in Fig. 4A, for two TG neurons recorded simul-

taneously, switching from isotonic solution to one which contained

no Ca2+-ions produced a slight reduction in the [Ca2+]i level. In the

absence of external Ca2+, an hypotonic solution did not produce a

[Ca2+]i signal, but re-application of the normal amount of Ca2+-ions

(2.4 mM), in the continuous presence of the hypotonic solution, led to

an immediate increase in [Ca2+]i with the usual time course for either

fast-type or slow-type responses. The very short delay in the [Ca2+]i

rise suggests that the in¯ux pathway was previously activated by the

hypotonic stimulus, and that this activation did not require the

presence of external Ca2+.

The possible role of intracellular Ca2+ stores in the response to cell

swelling was explored with cyclopiazonic acid (CPA) a blocker of

endoplasmic reticulum calcium pumps (Thomas & Hanley, 1994).

Prolonged application of CPA empties intracellular calcium stores in

TG neurons (our unpublished observations) as previously reported in

DRG neurons (Usachev & Thayer, 1999). After application of 20 mM

CPA, [Ca2+]i levels increased transiently, returning slowly

(t = 75 6 14 s) towards baseline (Fig. 4C). CPA was applied

for > 7 min to ensure an effective discharge of the stores. In the

continuous presence of CPA, application of 30% hypotonic solution

still elicited a signi®cant [Ca2+]i elevation. In fact, this elevation was

on average larger than in the absence of CPA, implying that

intracellular stores may act as a Ca2+ sink under normal conditions.

This increase in [Ca2+]i reversed upon return to the isotonic solution.

A summary of the results obtained in seven neurons is presented in

Fig. 4D. To further explore the possible role of intracellular calcium

stores in the response of TG neurons to cell swelling, they were

incubated for 1±3 h in isotonic solution containing 1 mM thapsigargin

and challenged soon thereafter (7±10 min) with 30% hypotonic

stimulation. Ten out of 19 (52%) TG neurons treated in this manner

responded to the hypotonic stimulus with a clear [Ca2+]i elevation,

averaging 147 6 47 nM above baseline (not shown). Altogether, the

results of removing external Ca2+, application of CPA, and incubation

in thapsigargin, suggest that intracellular Ca2+ stores are not essential

for stretch-evoked [Ca2+]i signals in TG neurons.

Effects of gadolinium and nickel on the hypotonicity-inducedincreases in [Ca2+]i

To explore the types of Ca2+ entry pathways involved in the [Ca2+]i

response to hypotonic stimulation, the effects of gadolinium and

nickel were tested. Gadolinium has been shown to block stretch-

activated channels in various cell types (Hamill & McBride, 1996),

and high concentrations of nickel block different types of voltage-

gated Ca2+-channels nonselectively (Zamponi et al., 1996).

The specimen recording in Fig. 5A illustrates that during applic-

ation of a 30% hypotonic solution, with the consequent increase in

[Ca2+]i, addition of 100 mM Gd3+ to the bathing solution sharply

reduces the [Ca2+]i concentration. As shown in C, this reduction

amounts, on average, to 71 6 3% after 3 min (n = 7). The blocking

effect of Gd3+ was already observed at 20 mM and it was only

partially reversible upon wash. Similar results were obtained when

Ni2+, at a concentration of 1 mM, was added to the hypotonic solution

(Fig. 5B). In this case, reductions in the [Ca2+]i rise after 3 min

exposure to Ni2+ were of a similar magnitude (see the averaged

results in C) as those obtained after 3 min exposure to 20±100 mM

Gd3+, indicating that both types of Ca2+-permeable channels

contribute to the swelling-activated Ca2+ in¯ow. In the case of Ni2+

the effects were completely reversible.

Coexistence of sensitivity to hypotonic stimulation andcapsaicin

Capsaicin is a rather speci®c excitatory substance for polymodal

nociceptors (Szolscayi, 1993). We investigated whether neurons

FIG. 5. Effects of blockers on hypotonicity-induced [Ca2+]i elevations. (A) Extracellularapplication of Gd3+ (100 mM), caused a markedreduction in the [Ca2+]i elevation triggered bya 30% hypotonic solution. (B) Effect of 1 mM

external Ni2+ upon the hypotonicity-induced[Ca2+]i response. Different neuron than in A.(C) Mean block of the hypotonicity-induced[Ca2+]i responses, 3 min after either 100 mM

Gd3+ (n = 11) or 1 mM Ni2+ (n = 12)application.

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responding to hypotonic solution with an increase in their [Ca2+]i

were also sensitive to capsaicin, and whether the time course of the

response was associated with a sensitivity or lack thereof to capsaicin.

A brief (20 s) bath application of 0.5 mM capsaicin gave rise to robust

increases in [Ca2+]i in 58% (n = 100) of the neurons tested (Fig. 6).

The average [Ca2+]i increase was 490 6 49 nM.

In the population of neurons tested with capsaicin, a 30%

hypotonic stimulus had been applied previously to de®ne their

mechanical responsiveness. Using both criteria (mechanical and

capsaicin sensitivity), we developed the dichotomous chart of TG

neuron classi®cation presented in Fig. 6. In this sample of 100

neurons, 80 reacted with an increase in their [Ca2+]i upon exposure to

hypotonic solutions. Of these 80 cells more than half, namely 49

cells, responded to capsaicin too, but the other 31 cells did not. As

detailed in the ®gure, capsaicin sensitivity as well as capsaicin

insensitivity could be found in both types of mechanically sensitive

neurons, fast-type and slow-type TG neurons. Of the remaining 20

neurons, insensitive to hypotonic stimulation, 11 were capsaicin-

insensitive, the other 9 gave responses to capsaicin. All six subgroups

of neurons displayed in Fig. 6 had very similar average cell diameters

(shown Fig. 6), suggesting that cell size is not a useful criterion to

classify neonatal TG neurons in culture.

Membrane potential changes evoked by hypotonic stimulation

To de®ne in more detail the mechanism of [Ca2+]i elevation upon cell

swelling, whole-cell patch-clamp recordings were combined with

[Ca2+]i measurements in TG neurons. The time course of the change

in membrane potential (upper panel) and [Ca2+]i response (lower

panel) upon mechanical stimulation is illustrated in Fig. 7A for a

typical experiment. The input resistance and excitability were tested

every 5 s with the application of rectangular negative and positive

current pulses (100 pA). During the control period the membrane

potential was stable and » ±64 mV. Hypotonic stimulation led to a

slow depolarization. Concurrently, [Ca2+]i increased (Fig. 7A, lower

trace), reaching a maximum at the end of the mechanical stimulation.

During the depolarization, the 100-pA pulse was able to elicit ®ring

of action potentials (Fig. 7B). Changes in membrane potential and

[Ca2+]i elevations were readily reversible upon return to isotonic

solution (Fig. 7C). On average (n = 5), cells depolarized from

±70 6 3 mV to ±44 6 8 mV during a 5-min period of 30%

FIG. 6. Capsaicin sensitivity and mechanical responsiveness de®ne six subpopulations of trigeminal neurons. Dichotomous key illustrating the different [Ca2+]i

response patterns observed in trigeminal neurons challenged with a 30% hypotonic stimulus and a brief 0.5 mM capsaicin application (always applied in thisorder). The percentage in the ®gure refers to the percentage of neurons classi®ed in each subgroup. For each subgroup the average diameter of neurons is alsoindicated (mean 6 SEM). The specimen records show the [Ca2+]i responses of typical neurons to a 5-min 30% hypotonic stimulus (left-hand column) and tocapsaicin (right-hand column). Further explanation in the text. The lowest [Ca2+]i calibration bar applies only to the lowest row of records.

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hypotonic stimulation. The depolarization induced by the hypotonic

stimulus was accompanied by an almost three-fold decrease in input

resistance, that changed from 410 6 155 MW in isotonic to

132 6 34 MW in 30% hypotonic solution (n = 4). The average

change in [Ca2+]i caused by the 30% hypotonic stimulus in the same

neurons is shown in Fig. 7D.

[Ca2+]i responses to hypotonic stimulation under voltage-clamp conditions

To determine if the hypotonically mediated [Ca2+]i elevations were

due exclusively to a depolarization of the neuron, simultaneous

measurements of whole-cell ionic current and [Ca2+]i were made.

Neurons were clamped at a potential of ±60 mV and membrane

conductance was monitored with a brief ±10-mV voltage step,

delivered every 5 s. As shown in Fig. 8A, upon hypotonic stimula-

tion, an inward holding current developed progressively, and this

holding current was accompanied by an increase in membrane

conductance. On average (n = 7), a holding current at ±60 mV

increased from ±7 6 14 pA to ±475 6 100 pA during hypotonic

stimulation. This inward current developed in parallel to an input

conductance increase from 2778 6 437 pS to 10796 6 1415 pS

(n = 7). The hypotonicity-induced changes in holding current were

accompanied by elevations in [Ca2+]i (Fig. 8A and C). In some

neurons, [Ca2+]i and conductance changes were maintained during

the stimulus while in others a strong degree of adaptation was

observed, with both parameters returning to basal values within the

time of stimulation (Fig. 8A).

Relation between electrophysiological characteristics and[Ca2+]i responses to hypotonic solutions

The electrophysiological properties of mice TG neurons, recorded

in vitro, are heterogeneous, both in neonatal (Cabanes et al., 1999)

and adult (LoÂpez de Armentia et al., 2000) animals. Neurons can be

divided into two main subgroups based on the shape of the action

potential recorded intracellularly (see also Belmonte & Gallego,

1983; Koerber et al., 1988; Waddell & Lawson, 1990). These two

subgroups could also be identi®ed in cultured TG neurons from

neonatal animals. As shown in Fig. 9A, some TG neurons had long

duration action potentials (2.8 6 0.2 ms, n = 41), with a marked

in¯exion, or hump, on their falling phase (type-S neurons). This

in¯exion was manifested as a second negative peak on the ®rst

derivative record of the action potential (Fig. 9A, lower trace). In

contrast, a second group of TG neurons (Fig. 9D) had shorter

duration (P < 0.01), monophasic action potentials (1.6 6 0.2 ms;

n = 7), and lacked an in¯exion on the falling phase of the spike

(type-F neurons). Other electrophysiological properties, in particular

time-dependent inward recti®cation (Mayer & Westbrook, 1983;

Scroogs et al., 1994), were also different in the two subgroups of TG

neurons. Time-dependent inward recti®cation was manifested by the

marked sag in the membrane potential trajectory during hyperpolar-

FIG. 7. Swelling depolarizes trigeminal neurons and elevates [Ca2+]i. (A) Time course of the changes in membrane potential (MP) (upper trace) and [Ca2+]i

(lower trace) in a trigeminal neuron during a 5-min 30% hypotonic stimulus. The points marked with the ®lled circles correspond to the specimen traces in B.(B) Membrane potential changes and responses to a 500-ms negative and positive intracellular current pulse (100 pA), applied every 5 s, in controlconditions, during the peak 30% hypotonic response and after return to isosmotic conditions. Records correspond to the time points marked black in A.(C) Average MP in control (isosmotic) conditions, in 30% hypotonic solution and after return to isosmotic solution. (D) Average [Ca2+]i in control (isosmotic)conditions, in 30% hypotonic solution and after return to isosmotic solution. Averages in C and D are from the same neurons (n = 4).

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izing current pulses. Figure 9B and E show the voltage responses to

negative current pulses in the type-S and type-F neurons shown in

Fig. 9A and D, respectively. Inward recti®cation, calculated as a

recti®cation index (see methods) was signi®cantly larger (P < 0.02)

in type-F neurons (33 6 4%) compared to type-S neurons

(24 6 1%). In seven additional cells, the presence or absence of a

hump was ambiguous (short hump) and they were not included in the

analysis.

We asked whether [Ca2+]i responses to hypotonic stimulation could

be different in type-S and type-F TG neurons. To perform this

analysis, intact neurons, loaded with fura2-AM, were exposed to a

30% hypotonic stimulus for 5 min and their [Ca2+]i response was

measured. Thereafter, the same neurons were approached with a

patch-pipette and recorded in the whole-cell con®guration, which

allowed the classi®cation into type-S and type-F neurons. Indeed,

[Ca2+]i responses to hypotonic stimuli in type-S neurons were

typically of the slow type (Fig. 9C). Thirty-two out of 38 (84%) type-

S neurons gave a [Ca2+]i response to the 30% hypotonic stimulus. The

average time to peak of the [Ca2+]i response was long (136 6 15 s)

and the mean [Ca2+]i amplitude was small (95 6 16 nM; n = 32). In

contrast, type-F neurons had generally faster [Ca2+]i responses, with a

time to peak of 83 6 21 s (n = 6), and much larger amplitudes

(381 6 135 nM) (P < 0.001). Six out of seven type-F neurons (86%)

responded to the hypotonic stimulation. The trace in Fig. 9F shows

the very fast and large [Ca2+]i response corresponding to the type-F

neuron illustrated in Fig. 9D.

Discussion

We have tested the effects of hypo-osmotic cell-swelling upon [Ca2+]i

in cultured mice TG neurons. The data show that extracellular

hypotonic solutions led to elevations in [Ca2+]i in a large fraction of

TG neurons. These [Ca2+]i elevations had a variable time course, their

amplitude encoded the intensity of the stimulus, required Ca2+ in¯ux

and were sensitive to blockers of mechanosensitive and voltage-gated

Ca2+ channels. Using simultaneous recordings of [Ca2+]i and whole-

cell ionic currents we found that hypo-osmotic cell-swelling led to a

signi®cant increase in membrane conductance, involving several

ionic mechanisms, including membrane potential depolarization and

voltage-independent Ca2+ in¯ux. Furthermore, cultured TG neurons

with brief action potentials and strong inward recti®cation showed

larger [Ca2+]i responses to hypo-osmotic stimulation. Previous work

has correlated these properties with mechanotransducing character-

istics of sensory terminals (Belmonte & Gallego, 1983; Koerber et al.,

1988).

Origin of the [Ca2+]i responses to hypo-osmotic solutions inTG neurons

Changes in cell volume produced by extracellular application of

hypo- and hyper-osmotic solutions are known to evoke [Ca2+]i

elevations in neurons involved in osmoreception (Oliet & Bourque,

1993) and in aortic baroreceptor neurons (Cunningham et al., 1995)

as well as in various types of non-neural cells (Kim & Fu, 1993;

Harada et al., 1994; Chen et al., 1996). The mechanisms involved in

FIG. 8. Mechanical stimulation activates an inward current in TG neurons. (A) Time course of the changes in holding current (open circles), conductance(®lled triangles) and [Ca2+]i (open triangles, lower diagram) in a trigeminal neuron during a 5-min 30% hypotonic stimulus. (B) Average current at a holdingpotential of ±60 mV in control (isosmotic) conditions, in 30% hypotonic solution (hypo) and after return to isosmotic solution (wash). (C) Average [Ca2+]i incontrol (isosmotic) conditions, in 30% hypotonic solution and after return to isosmotic solution. Averages in B and C are from the same neurons (n = 7).

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[Ca2+]i elevation during cell swelling are diverse in the various cell-

types examined (Foskett, 1984). These mechanisms include activ-

ation of Ca2+ in¯ux and intracellular Ca2+ release. Few studies have

characterized responses to hypo-osmotic cell-swelling in neurons

(Leaney et al., 1997; Aitken et al., 1998). The simplest explanation

for our results suggests the involvement of at least two distinct

mechanisms, with different voltage-dependence and time course (see

below) in the [Ca2+]i increase.

Transduction of mechanical forces by the peripheral endings of

mammalian primary sensory neurons is thought to be mediated by

opening of stretch-activated channels (SACs) (French, 1992;

Drummond et al., 1998). Many SACs are permeable to calcium

(Yang & Sachs, 1990). Albeit speculative at this stage, the response

to hypo-osmotic swelling may be interpreted within the framework of

the known mechanosensitivity of a large fraction of trigeminal ®bers

in vivo. Thus, [Ca2+]i elevations induced by hypotonic solutions took

place in only a fraction of TG neurons, required Ca2+ in¯ux, exhibited

a component of the response that was voltage-independent and were

greatly reduced by gadolinium, a generic blocker of many SACs

(Hamill & McBride, 1996). The Ca2+ response that appeared most

closely related to opening of mechanosensitive, Ca2+-permeable

channels, was the one with a fast time course, that coincided with the

time of maximal rate of swelling. Fast calcium responses were also

typical of type-F TG neurons. Our intracellular studies in the TG of

mice in vitro (LoÂpez de Armentia et al., 2000) indicate that type-F

neurons with strong inward recti®cation are mostly Ad neurons, the

CV range typical of most TG mechanosensory ®bers. In the petrosal

ganglion, 64% of myelinated baroreceptor neurons had fast/narrow

spikes, while 100% of the chemosensitive neurons had slow/humped

spikes (Belmonte & Gallego, 1983). An association between narrow

spikes, large inward recti®cation and strong mechanosensitivity has

also been reported in cat DRG neurons (Koerber et al., 1988).

Is there any other evidence in the literature that correlates the

mechanosensitive phenotype of peripheral neurons with [Ca2+]i

responses to hypo-osmotic cell swelling? It is interesting to note

that, in cultured sympathetic neurons, which are not known to posses

mechanosensitivity, hypotonic stimulation up to 40% did not cause a

rise in [Ca2+]i (Leaney et al., 1997). In the same neurons McCarter

et al. (1999) did not ®nd a whole-cell current activated by mechanical

deformation of the soma, unlike results obtained by the same authors

in the DRG.

In parallel to the increase in [Ca2+]i by hypo-osmotic stimulation, a

membrane potential depolarization was observed. Application of

1 mM Ni2+, a nonspeci®c blocker of voltage-gated Ca2+ channels,

reduced the [Ca2+]i rise, suggesting that opening of voltage-gated

channels may additionally participate in the swelling-induced [Ca2+]i

responses. Moreover, activation of calcium in¯ux and activation of

chloride currents during hypo-osmotic cell swelling have been

described in a variety of cells (reviewed by Foskett, 1994; Nilius

et al., 1996), including neurons (Leaney et al., 1997) and may also

contribute to membrane depolarization. Ion substitution experiments

indicate that a fraction of the hypotonically induced inward current

observed in our experiments was carried by chloride ions (our

unpublished observations). Our results here, and the recent discovery

of a mechanosensitive chloride channel in growth cones of cultured

rat DRG neurons (Imai et al. 2000) strengthens the implication of

FIG. 9. Correlation between electrophysiological properties and [Ca2+]i responses to hypotonic stimulation. (A) Action potential (upper trace) and timederivative (lower trace) evoked in a type-S trigeminal neuron by a depolarizing current pulse at threshold. Note the marked in¯exion during the falling phaseof the action potential. The dotted line marks 0 mV. (B) Membrane voltage responses to hyperpolarizing current pulses (current steps, 15 pA) in the sameneuron as in A. (C) Time course of the increase in [Ca2+]i of the same neuron in response to a 5-min application of a 30% hypotonic test solution. (D)Action potential (upper trace) and time derivative (lower trace) evoked in a type-F trigeminal neuron by a depolarizing current pulse. Note the brief actionpotential without an in¯exion on its falling phase. (E) Membrane voltage responses to hyperpolarizing current pulses (current steps, 10 pA) in the sameneuron as D. Note the marked sag in the voltage response at the more hyperpolarized levels. (F) Time course of the [Ca2+]i increase in response to a 5-minapplication of a 30% hypotonic test solution. Same neuron as D. Calibration bars in A±C also apply to the corresponding traces in D±F.

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anion conductances in mechanotransduction, previously advanced in

nonexcitable cells.

Finally, although our data with zero Ca2+ and SERCA pump

blockers (CPA and thapsigargin) show that entrance of extracellular

Ca2+ is the major source of the [Ca2+]i response to hypo-osmotic

stimulation in TG neurons, the time course and characteristics of the

transient [Ca2+]i response are compatible with the possible involve-

ment of a calcium-induced calcium release (CICR) mechanism in

augmenting the [Ca2+]i elevation. Such a mechanism has been

identi®ed in a subpopulation of DRG neurons (Shmigol et al., 1995;

Usachev & Thayer, 1999). In heart myocytes, this process implies

that the initial Ca2+ in¯ux evoked by membrane stretch will often

lead to successive waves of calcium-induced calcium release

(Sigurdson et al., 1992)

Functional heterogeneity of cultured TG neurons

Mechanosensory neurons in sensory ganglia have been classi®ed as

low threshold neurons, that are maximally activated by low-intensity

forces, and as high-threshold, nociceptive neurons that are preferen-

tially excited by noxious stimuli (Burgess & Perl, 1973). Neurons that

respond additionally to exogenous irritants, endogenous chemicals and

extreme temperatures were categorized as polymodal nociceptive

neurons. About 80% of cultured TG neurons responded with a [Ca2+]i

rise to exposure to 30% hypotonic solution, a stimulus that invariably

caused cell swelling (Fig. 2) and is expected to cause a de®nite stretch

of the cell membrane and thus to activate most if not all of those TG

neurons possesing mechanosensitivity. This proportion roughly cor-

responds with the total population of sensory ®bers responding to

mechanical forces (low- and high-threshold mechanosensory axons,

polymodal nociceptor axons) in the saphenous nerve of the rat that

innervates the hind limb skin (Lynn & Carpenter, 1982).

In the present experiments, the magnitude of the [Ca2+]i rise and

the percentage of responding neurons were dependent on the intensity

of the stimulus, suggesting differences in threshold among individual

TG neurons. A small fraction of them displayed [Ca2+]i responses of

large amplitude, rapid time course and shorter duration, that

desensitized upon repeated stimulation. These neurons showed a

short-duration AP devoid of a hump and prominent inward recti®c-

ation. Neurons with similar electrophysiological properties have been

recorded in newborn and adult DRG, and in TG superfused in vitro

which showed conduction velocities in the myelinated range (Zhang

et al., 1998; Cabanes et al., 1999; LoÂpez de Armentia et al., 2000). It

is tempting to speculate that the group of purely mechanosensitive,

F-type TG neurons (12%), with fast and prominent [Ca2+]i responses

found in the present experiments, correspond to low-threshold

mechanosensory neurons with highly sensitive, phasic responses to

peripheral mechanical stimulation and a high conduction velocity

(Zucker & Welker, 1969; Lynn & Carpenter, 1982).

The mechanisms underlying the very different time courses and

amplitudes of the calcium response in subsets of TG of neurons were

not investigated directly. An obvious hypothesis is that the calcium

response depends on the density and/or biophysical properties of

channels in the cell membrane. Our results ruled out any direct

relation to differences in the degree or kinetics of cell volume

regulation (Table 2). The calcium response was not related to marked

differences in cell size either. This was not surprising in light of

previous studies. Thus, a considerable overlap in cell size has been

found between functionally chraracterized populations of primary

sensory neurons (see Waddel & Lawson, 1990), especially within the

C and Ad conduction velocity groups (typical for TG neurons),

making cell size an unreliable parameter for classi®cation purposes in

the TG ganglion. The calcium response was also unrelated to the TG

neuron's ability to ®re tonically or phasically upon membrane

depolarization (not shown). It is possible that differences in their Ca2+

buffering capacities may play a role or differences in the activation

and inactivation properties of the Ca2+ in¯ux pathway.

[Ca2+]i responses to hypo-osmotic swelling were observed in

capsaicin-sensitive and capsaicin-insensitive neurons. This ®nding is

consistent with the mechanical sensitivity of some types of

nociceptors, known as polymodal receptors (Bessou & Perl, 1969;

Lynn & Carpenter, 1982). Thus, it is well established that many heat-

sensitive nociceptive ®bers in the trigeminal nerve are also sensitive

to mechanical stimuli (Belmonte & Giraldez, 1981; Gallar et al.,

1993; MacIver & Tanelian, 1993), i.e. they are polymodal (see

above).

Neurons unresponsive to hypo-osmotic stimuli and with small or

undetectable responses to capsaicin stimulation (11% in our sample)

may represent cold receptors. Four percent of the cutaneous ®bers

innervating the rat hind limb (Lynn & Carpenter, 1982), and

presumably a higher proportion of trigeminal sensory ®bers, are

cold-sensitive (Kenshalo et al., 1976).

Note added in proof

After submission of this manuscript, two groups have reported the

cloning mouse of a non-selective cation channel activated by cell

swelling (Strotmann et al., 2000; Liedtke et al., 2000).

Acknowledgements

The authors are grateful to Rosa Velasco, Eva Quintero and Alfonso PeÂrez fortheir excellent technical assistance and to Stuart Ingham for illustrations. Wethank Dr Roberto Gallego and Dr Miguel Valdeolmillos for helpful commentsand suggestions on the manuscript. This work was supported by funds from theSpanish Ministerio de EducacioÂn y Cultura (SAF99-0066-C02-01) and a grantfrom Biomatrix, Inc., Ridge®eld, New Jersey, USA. F.V. is supported by theSpanish Council for Scienti®c Research (CSIC).

Abbreviations

CPA, cyclopiazonic acid; DRG, dorsal root ganglion; F, ¯uorescence; fura2-AM, acetoxymethylester form of fura-2; Keff, effective binding constant; NGF,nerve growth factor; p, osmotic pressure; R, ratio (of ¯uorescence); RI,recti®cation index; RVD, regulatory volume decrease; SACs, stretch-activatedchannels; TG, trigeminal ganglion; Type-F (neurons), have short durationmonophasic action potentials lacking an in¯exion on the falling phase; Type-S(neurons), have long duration action potentials with a marked in¯exion, orhump, on the falling phase ; V, volume; Vm, membrane potential.

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