Neurons with distinctive firing patterns, morphology and distribution in laminae V–VII of the...

Neurons with distinctive firing patterns, morphology anddistribution in laminae V–VII of the neonatal rat lumbarspinal cord

Peter Szucs, Francis Odeh, Karolina Szokol and Miklos AntalDepartment of Anatomy, Histology and Embryology, Faculty of Medicine, Medical & Health Science Centre, University of Debrecen,Debrecen, H-4012 Hungary

Keywords: biocytin labelling, slice preparation, spinal interneurons, whole cell patch clamp

Abstract

It is generally accepted that neurons in the ventral spinal grey matter, a substantial proportion of which can be regarded as constituentsof the spinal motor apparatus, receive and integrate synaptic inputs arising from various peripheral, spinal and supraspinal sources.Thus, a profound knowledge concerning the integrative properties of interneurons in the spinal ventral grey matter appears to beessential for a fair understanding of operational principles of spinal motor neural assemblies. Using the whole cell patch clampconfiguration in a correlative physiological and morphological experimental approach, here we demonstrate that the intrinsic membraneproperties of neurons vary widely in laminae V–VII of the ventral grey matter of the neonatal rat lumbar spinal cord. Based on their firingpatterns in response to depolarizing current steps, we have classified the recorded neurons into four categories: ‘phasic’, ‘repetitive’,‘single’ and ‘slow’. Neurons with firing properties characteristic of the ‘phasic’, ‘repetitive’ and ‘single’ cells have previously beenreported also in the superficial and deep spinal dorsal horn, but this is the first account in the literature in which ‘slow’ neurons have beenrecovered and described in the spinal cord. The physiological heterogeneity in conjunction with the morphological correlation anddistribution of neurons argues that different components of motor neural assemblies in the spinal ventral grey matter possess differentsignal processing characteristics.

Introduction

There is general agreement that neural assemblies that are responsible

for motor rhythm and pattern generation, and thus for the initiation of

rhythmic alternating motor behaviours like locomotion are confined to

the ventral aspects of the spinal grey matter (Szekely, 1963; Pratt &

Jordan, 1987; Schmidt et al., 1988; Pearson, 1993; Kjaerulff & Kiehn,

1996). It is also established that spinal motor neural circuits constitute

a functionally heterogeneous population of neurons. It has been shown

that some of these cells contribute to networks generating pacemaker

like activity (Ho & O’Donovan, 1993; Kjaerulff & Kiehn, 1996), while

others retain mechanisms that render a well-balanced spatial and/or

temporal integration of synaptic inputs arising from various sources

possible (Harrison & Jankowska, 1985; Morisset & Nagy, 1998;

Garraway & Hochman, 2001; Nakatsuka et al., 2002). For instance,

in addition to a strong drive from the spinal motor rhythm and pattern

generating circuits, last-order premotor interneurons, neurons that

conduct nerve signals to motoneurons, may also receive sensory inputs

and commands from various supraspinal motor centres (Moschovakis

et al., 1991; Jankowska, 1992; Degtyarenko et al., 1996, 1998). Thus,

they have crucial importance in the integration of activities generated

by the spinal motor apparatus, sensory information and nerve impulses

descending from voluntary and nonvoluntary higher motor centres.

Although the sources of synaptic inputs that are received by other

elements of the spinal motor neural assembly may vary in a wide range,

it is quite possible that similar to last-order premotor interneurons,

most, if not all, of the neurons in this circuitry receive multiple inputs.

The integration of various synaptic inputs on the somato-dendritic

membrane depends on several factors including the intrinsic passive

and active membrane properties as well as the morphology of the

neurons. Therefore extensive morphological studies in the ventral

aspect of the spinal grey matter has been performed (Kjaerulff et al.,

1994; Moschovakis et al., 1992; Puskar & Antal, 1997). The input

properties and the participation of some ventral horn neurons in various

reflex pathways have also been characterized (Brink et al., 1983;

Edgely & Jankowska, 1987; Wheatley et al., 1994; Jonas et al., 1998).

However, our present comprehension concerning the active and pas-

sive membrane properties of these neurons is limited, although a

profound knowledge of this matter appears to be a prerequisite for

a fair understanding of fundamental operational characteristics of

spinal neural assemblies in laminae V–VII.

Accordingly, in the experiments presented here we investigated the

firing patterns of neurons in response to suprathreshold depolarizing

current pulses in laminae V–VII of the neonatal rat lumbar spinal cord.

We have also made an attempt to find some correlation among the

physiological and morphological properties as well as the location of

the investigated neurons. Preliminary observations from this study

have been reported in abstract form (Szucs et al., 2002).

European Journal of Neuroscience, Vol. 17, pp. 537–544, 2003 � Federation of European Neuroscience Societies

doi:10.1046/j.1460-9568.2003.02484.x

Correspondence: Dr Peter Szucs, as above.

E-mail: [email protected]

Received 12 August 2002, revised 26 November 2002, accepted 2 December 2002

Materials and methods

Preparation of spinal cord slices

Experiments were carried out on new-born and young (P0–8) Wistar–

Kyoto rats (Godollo, Hungary). All animal study protocols were

approved by the Animal Care and Protection Committee at the

University of Debrecen, Hungary, and were carried out in accordance

with the European Communities Council Directives. Under deep

isofluorane anaesthesia, the animals were decapitated and the lumbar

spinal cord was dissected in an ice cold artificial cerebrospinal fluid

(ACSF, pH 7.4) that contained (in mM): NaCl, 130; NaHCO3, 24; KCl,

3.5; NaH2PO4, 1.25; CaCl2, 1; MgSO4, 3; and glucose, 10, and was

saturated with 95% O2 and 5% CO2. After removing the meninges,

blocks of the lumbar spinal cord were embedded into agar and

sectioned at 300–400 mm on a Vibratome. Slices were incubated in

ACSF at room temperature for at least 1 h prior to recording.

Electrophysiological recordings

Slices were transferred into a recording chamber, which was constantly

perfused with oxygenated ACSF. Neurons located in laminae V–VII

were visually identified with a Zeiss Axioskop FS microscope

equipped with a �40 water immersion objective, DIC filter and an

infrared CCD camera system (Hamamatsu, Japan). Conventional

whole-cell patch clamp recordings were performed in current clamp

mode (analogous to bridge mode of conventional microelectrode

amplifiers) using an Axopatch 1D amplifier (Axon Instruments, Union

City, CA, USA) and pipettes with a resistance of 4–7 MO. The

electrode filling solution contained (in mM): K-gluconate, 126;

KCl, 4; ATP, 4; GTP, 0.3; Phosphocreatine, 10; Hepes, 10; and

0.5% biocytin (Sigma, St. Louis, MO, USA) at pH 7.2. To prevent

spontaneous firing, which is not unusual in slice preparations due to

either electrode penetration or disruption of neuronal circuitry during

slicing, the membrane potential was kept constant in the range between

�60 mV and �65 mV throughout the experiment with a tonic hyper-

polarizing DC current injection in the range of 0–100 pA, a commonly

used method (Maccaferri et al., 2000). If the DC current needed to

keep the cells stable increased above the given range during the

experiment, the recording was discarded from the study. Firing

patterns were obtained by applying 800 ms long incrementing current

steps ranging from �30 pA to 100 pA. Capacitance and series resis-

tance were not compensated. All recordings were performed at room

temperature.

Data acquisition and analysis

Data were recorded on an IBM PC, filtered at 5 kHz, digitized with a

Digidata 1320 A A/D board and analyzed by using pClamp (Axon

Instruments, Union City, CA, USA), Origin (Microcal Software,

Northampton, MA, USA) and Whole Cell Program and Electrophy-

siology Data Recorder (courtesy of Dr J. Dempster, University of

Strathclyde, UK) software packages.

From firing patterns evoked by the application of suprathreshold

current steps (100 pA, 800 ms) the following parameters were ana-

lyzed: spike frequency, interspike intervals (ISI), ratio between the

amplitudes of the first and last spikes.

Membrane time constant (t) was determined by fitting one decaying

exponential function to the cells’ voltage response to small negative

current steps (�10 pA, 800 ms). Current–voltage (I–V) curves were

constructed by plotting the amplitude of the membrane potential

change against the amplitude of the applied subthreshold current step.

Action potential and afterhyperpolarization (AHP) amplitudes were

measured from action potential threshold. The half-width of the action

potentials was determined at half peak amplitude measured from

threshold. Action potential threshold and the shape of the AHP was

evaluated at the lowest current step that generated action potentials

from resting membrane potential (Ruscheweyh & Sandkuhler, 2002).

Histological processing and morphological evaluation

Following the recording session, the slices were placed between two

Millipore filters (Millipore Corporation, Bedford, MA, USA) to avoid

deformation and transferred into a fixative containing 4% paraformal-

dehyde, 1.25% glutaraldehyde and 0.2% picric acid in 0.1 M phosphate

buffer (pH 7.4) for 1–4 days. Slices were sectioned at 60mm on a

Vibratome. To visualize biocytin that diffused from the patch pipette

into the recorded neuron, sections were treated according to the avidin-

biotinylated horseradish peroxidase method (Extravidin, diluted

1 : 1000, Vector Laboratories, Burlingame, CA, USA) and the histo-

chemical reaction was completed with a diaminobenzidine (Sigma, St.

Louis, MO, USA) chromogen reaction. Sections were counterstained

with toluidine blue, dehydrated and mounted with DPX (Fluka, Buchs,

Switzerland).

The somata as well as the dendritic and axonal arbors of the recorded

and labelled neurons were recovered and three dimensionally recon-

structed from serial sections using a computer aided NEUROLUCIDA

reconstruction system (Microbrightfield Inc., Villiston, VT, USA)

installed onto a Leitz Laborlux microscope. To define the laminar

location of the recorded and labelled cells in the spinal grey matter we

have applied the segmentation scheme of Kjaerulff et al., (1994) that

they constructed specifically for the neonatal rat lumbar spinal cord.

The maximum cross-sectional area as well as the minimum and

maximum diameters of the somata were measured. The ratio of the

maximum and minimum diameters (aspect ratio) was calculated.

Dendrograms of the labelled neurons were constructed, and the

numbers of stem dendrites and branch points along the dendritic trees

in relation to the distance from the cell body were counted. No variance

analysis of the dendritic and axonal distribution was performed inside

or between the different cell groups.

All curve fits were performed using the built-in iterative Levenberg–

Marquadt algorithm in ClampFit (Axon Instruments, Union City, CA,

USA). All numerical data are presented as mean� SEM. Statistical

significance was assessed using Student’s t-test.

Results

Seventy neurons were recorded. In certain cases more than one cell

was recorded in the same slice. Both the electrophysiological record-

ings and the biocytin labelling were successful in 47 cases. Conse-

quently, data presented in this study are based on the results obtained

from these 47 cells. The average resting membrane potential of the

recorded cells were �51.71� 1.52 mV. On the basis of their firing

patterns evoked by suprathreshold depolarizing current pulses the

recorded neurons were divided into four groups that we classified

as: (i) ‘phasic’ neurons (10 cells); (ii) ‘repetitive’ neurons (15 cells);

(iii) ‘single’ neurons (9 cells); and (iv) ‘slow’ neurons (8 cells). Five

neurons could not be classified according to this scheme; their firing

patterns showed similarities partly to the ‘phasic’ partly to the ‘repe-

titive’ neurons.

Firing patterns

‘Phasic’ neurons discharged a continuous train of action potentials

during suprathreshold depolarizing current pulses (Fig. 1A). In addi-

tion, they showed a marked spike accommodation including a pro-

gressive increase in interspike intervals and attenuation of spike

amplitude during the 800 ms long depolarizing current step (Fig. 1B).

Short duration monophasic AHPs were also observed after the spikes

538 P. Szucs et al.

� 2003 Federation of European Neuroscience Societies, European Journal of Neuroscience, 17, 537–544

(Fig. 1C). The average ratios between the first and last spike ampli-

tude and interspike interval were 5.56� 0.72 and 0.44� 0.03, respec-

tively.

Similar to ‘phasic’ cells, ‘repetitive’ neurons also discharged a

continuous train of action potentials during suprathreshold depolariz-

ing current pulses (Fig. 1A). In contrast to the ‘phasic’ cells, however,

we did not observe any obvious sign of spike accommodation or

attenuation (Fig. 1B), but spikes were always followed by a marked

slow monophasic AHP (Fig. 1C).

In contrast to all of the other cell types, ‘single’ neurons fired a

single (in some cases two) action potential upon suprathreshold

depolarization (Fig. 1A). Consecutive spikes could be evoked only

after repolarization to resting membrane potential. In these cells only a

small AHP could be detected after the single spike (Fig. 1C).

Similar to the ‘repetitive’ cells, ‘slow’ neurons also discharged a

continuous train of action potentials during suprathreshold depolar-

ization (Fig. 1A). There was no obvious sign of spike accommodation

or attenuation, and spikes were always followed by a long-lasting

Fig. 1 Firing patterns of recorded neurons in laminae V–VII. (Column A) Traces show the voltage response to the current steps indicated below. The small inserts(Column B) demonstrate the relationship between the consecutive action potential amplitudes. ‘Phasic’ neurons showed a marked accommodation of both spikeamplitude and interspike intervals. ‘Repetitive’ neurons maintained their spike amplitudes and frequency during the suprathreshold depolarization induced dischargeperiod. ‘Single’ neurons fired only one action potential. ‘Slow’ neurons showed a low spike frequency but no obvious sign of spike accommodation or attenuation.(Column C) Enlarged initial spikes of a neuron from each firing pattern group. Arrowheads indicate the AHPs following the spikes.


Interneurons in laminae V–VII of the spinal cord 539

monophasic AHP (Fig. 1B and C). However, the average spike fre-

quency of ‘slow’ neurons (8.9� 0.6Hz) was significantly lower than

that of the ‘repetitive’ cells (21.83� 1.5Hz) (P< 0.001).

Passive and active membrane parameters

Although the resting membrane potential and input time constant

tended to be smaller in ‘slow’ and ‘single’ neurons than in ‘phasic’ and

‘repetitive’ cells, statistical analysis revealed that the values of resting

membrane potentials, input time constants, action potential overshoots

and half-widths did not differ significantly among the neurons with

different firing patterns. The smallest average input time constant was

observed in the ‘single’ group (26.6� 8.82 ms). Also, ‘phasic’ and

‘single’ cells showed a tendency for wider action potentials than that of

the other two firing pattern groups, although a statistically significant

difference could not be established (Table 1).

However, certain parameters made the ‘single’ and ‘slow’ cells

unique and obviously different from the ‘phasic’ and ‘repetitive’

neurons. The amplitudes of action potentials were similar in ‘phasic’,

‘repetitive’ and ‘slow’ neurons. ‘Single’ cells presented action poten-

tials with the smallest amplitude (54.44� 4.14 mV) and overshoot

(16.84� 6.16 mV) but with the highest threshold (�40.94� 5.95 mV)

among all types of neurons. Action potential thresholds were similar in

the ‘phasic’ and ‘repetitive’ group, while ‘slow’ neurons had the lowest

threshold values (�45.2� 1.47). Similarly to the ‘phasic’ and ‘repe-

titive’ cells, ‘slow’ neurons presented a strong afterhyperpolarization,

while the average amplitude of AHP was significantly smaller in the

‘single’ cell population (Table 1). The current–voltage curves appeared

to be flat in ‘slow’ and ‘single’ neurons, while they were steeper in

‘repetitive’ and steepest in the ‘phasic’ cells (Fig. 2), indicating that the

input resistance of ‘slow’ and ‘single’ cells is lower than that of

neurons in the ‘repetitive’ and especially in the ‘phasic’ groups. Indeed

‘slow’ cells presented the smallest input resistance among the recorded

neurons (Table 1).

Laminar distribution and morphometric parameters

Of the 47 reconstructed neurons, 13 were located within laminae V–VI

and 34 in lamina VII. Similarly to membrane parameters, the dis-

tribution and morphology of ‘phasic’ and ‘repetitive’ neurons were

similar to each other, while ‘single’ and ‘slow’ cells showed unique

characteristics.

Most of the ‘phasic’ (8 out of 10) and ‘repetitive’ (13 out of 15)

neurons presented multipolar perikarya with 4–6 primary dendrites,

and were distributed equally in laminae V–VII (Fig. 3). Of the nine

‘single’ neurons seven were recovered in the lateral aspect of laminae

V–VI, and only two were located in lamina VII. In these seven neurons

the small multipolar perikarya gave rise to 3–5 stem dendrites, and

presented an extensive but poorly arborizing dendritic tree with a

preferred orientation in the dorso-ventral direction (Fig. 3). In five cells

out of the seven, the axon was also labelled. After arising from the cell

body the axons turned ventrally and terminated in the ventral grey

matter. From the neurons investigated in this study, ‘slow’ neurons

presented the largest and most elongated perikarya. Six out of the eight

recovered ‘slow’ neurons had 6–8 primary dendrites, were arranged in

two groups that arose from the opposite poles of the elongated somata

Fig. 2 Current–voltage relations of neurons in laminae V–VII. The graph showsthe amplitude of the membrane voltage change plotted against the appliedcurrent step. All numerical data are presented as mean� SEM.

Table 1. Passive and active membrane parameters of neurons in laminae V–VII

Neuron type

Phasic(n¼ 9)

Repetitive(n¼ 14)

Slow(n¼ 7)

Single(n¼ 8)

Significance(Student’s t-test)

Resting membrane potential (mV) �51� 4.2 �53.6� 2.54 �49� 2.79 �47� 3.97 NSInput time constant (ms) 50.83� 7.74 41.81� 5.4 32.36� 4.42 26.6� 8.82 NSInput resistance (MW) 994� 197 822� 151 304� 59 643� 166 Phasic vs. slow, P< 0.01;

repetitive vs. slow, P< 0.01;phasic vs. single, P< 0.05.

Action potential threshold (mV) �43.8� 2.05 �43.7� 3.17 �45.2� 1.47 �40.94� 5.95 NSAction potential amplitude (mV) 69� 4.48 65.79� 2.57 69.03� 2.3 54.44� 4.14 Phasic vs. single, P< 0.05;

repetitive vs. single, P< 0.05;slow vs. single, P< 0.01.

Action potential overshoot (mV) 25.15� 5.28 23.97� 3.95 23.42� 4.06 16.84� 6.16 NSAction potential half-width (ms) 1.41� 0.17 1.08� 0.08 1.1� 0.07 1.52� 0.2 NSAHP amplitude (mV) 13.26� 2.49 16.98� 2.3 14.79� 2.16 8.91� 1.35 Repetitive vs. single, P< 0.01;

slow vs. single, P< 0.05.

Average spike frequency (Hz) NA 21.83� 1.5 8.9� 0.6 NA Repetitive vs. slow, P< 0.001.

AHP, afterhyperpolarization; NA, not applicable; NS, no significant differences.


540 P. Szucs et al.

and formed a richly arborizing bushy dendritic arbor. In contrast to the

other cells, ‘slow’ neurons were confined to the ventromedial part of

lamina VII (Fig. 3). In the case of seven neurons the axon was also

labelled. The labelled axon arose from the cell body and extended

either towards the midline or turned laterally. Of the five medially

orientated axons, four crossed the midline in the anterior commissure

and terminated in the contralateral ventral grey matter. The laterally

orientated axons targeted the lateral motor column.

Measuring the cross-sectional areas as well as the largest and

smallest diameters of stained perikarya it turned out that the shape

and size of cell bodies of ‘phasic’, ‘repetitive’ and ‘single’ neurons

were very similar (Table 2). According to these parameters, however,

the perikarya of ‘slow’ neurons turned out to be different from the

others. They presented a significantly larger and apparently more

elongated perikarya than neurons in the other groups (Table 2).

Analysis of branch point distribution histograms obtained from

dendrograms of neurons with different firing patterns showed that

in this regard the dendritic arborization pattern of ‘phasic’, ‘repetitive’

and ‘single’ neurons were quite similar to each other. However, ‘slow’

neurons presented dendritic structures that were substantially different

from the dendritic arbores of cells in the other groups. The long,

slender dendrites of ‘phasic’, ‘repetitive’ and ‘single’ neurons

Fig. 3 Photomicrograph and Neurolucida reconstruction of representative samples of ‘phasic’, ‘repetitive’, ‘single’ and ‘slow’ neurons. Cell bodies and dendrites areshown in black, while the axons are grey. Dots on the schematic representations of the neonatal spinal cord slice adopted from Kjaerullf et al. (1994) indicate the locationof successfully recorded and labelled neurons in each group. On the drawings, the borders between the grey and white matter are drawn with dashed lines, whereascontinuous grey lines represent the borders of Rexed laminae. Roman numbers identify Rexed laminae V–VI and VII. D, dorsal; M, medial. Scale bars, 100mm.



branched infrequently, in a way that most of the branching points were

scattered within a distance of 100–150 mm from the soma (Fig. 4). In

the ‘phasic’ and ‘single’ groups, one or two primary dendrites occa-

sionally extended away from the cell body in a way that they gave

branches only in a distance of 300–600mm from the soma. This distant

accumulation of branching points presented a second smaller hump on

the branch point distribution histograms (Fig. 4). In contrast to this,

‘slow’ neurons formed a richly arborizing bushy dendritic arbor in

which the dendrites continuously branched as they receded away from

the cell body (Fig. 4).

Discussion

In this study we have documented that intrinsic membrane properties

as well as the morphology of neurons vary widely in the ventral

grey matter of the neonatal rat lumbar spinal cord. Based on their

firing patterns in response to depolarizing current steps, we have

classified the recorded neurons into four categories, and distinguished

‘phasic’, ‘repetitive’, ‘single’ and ‘slow’ neurons. Neurons with firing

properties characteristic of the ‘phasic’, ‘repetitive’ and ‘single’ cells

have previously been reported in the superficial and deep dorsal

horn of the spinal cord (Hochman et al., 1997; Prescott & Koninck,

2002) as well as among cultured spinal neurons (Jo et al., 1998).

However, this is the first account in the literature in which ‘slow’

neurons have been recovered and described in the spinal cord. Al-

though the input resistance values of the neurons in our sample were

slightly different from the values described in the above studies, other

authors report similar data especially in young animals (Hochman

et al., 1997).

We have frequently observed spontaneous excitatory and inhibitory

postsynaptic potentials throughout the recordings. Although this

spontaneous activity in the network might modify the discharge

patterns of the recorded neurons, it is likely that this external influence

had little, if any effect on our results, as ‘phasic, ‘repetitive’ and

‘single’ neurons have previously been identified also among cultured

spinal neuron (Jo et al., 1998), where this network effect can obviously

be excluded. Thus we assume that the different discharge patterns of

neurons that we have demonstrated here are primarily determined by

the intrinsic membrane properties of the recorded neurons.

‘Phasic’ and ‘repetitive’ neurons

The separation of ‘phasic’ and ‘repetitive’ neurons into two distinct

categories might be artificial. They may only represent the two

extremes of one heterogeneous cell population. The fact that in our

sample there were five neurons that could not be classified according to

this scheme, as their firing patterns showed similarities partly to the

‘phasic’ partly to the ‘repetitive’ neurons argues in favour of this

notion. All of the ‘phasic’, ‘repetitive’ and unclassified neurons show a

continuous high frequency spiking to suprathreshold stimulation,

where spikes are followed by AHPs with similar amplitude. This

AHP is likely to be responsible for the spike frequency accommodation

in the case of ‘phasic’ cells. The mechanism behind the decreasing

spike amplitude in ‘phasic’ neurons can be either deactivation of

sodium channels and/or a raised spike threshold level. One could also

argue that transient electrode blockage during the depolarizing current

step could result in a similar spike amplitude decrease. However, the

random occurrence/development of such electrode blockage during

the course of the experiment would also change the firing pattern of

neurons in all the other groups; this was not observed in our experi-

ments. Furthermore, this typical firing pattern was also reported earlier

by others in cells with membrane properties similar to the neurons in

our ‘phasic’ group (Hochman et al., 1997; Jo et al., 1998).

Paradoxically neurons with a characteristic ‘repetitive’ discharge

pattern, present slightly larger and markedly longer AHPs, without

obvious signs of spike accommodation. It is possible that the more

developed AHP may allow a more effective de-inactivation of sodium

channels, and therefore ensures the uniform amplitudes and threshold

levels of spikes in case of the ‘repetitive’ cells (Thomson et al., 1989).

Such a mechanism requires further investigation.

Neurons with the ‘phasic’ and ‘repetitive’ properties are scattered all

over the entire cross-sectional area of the spinal grey matter. In

addition to laminae V–VII, where we located them in the present

study, neurons with identical firing patterns were identified also in the

superficial and deep spinal dorsal horn (Hochman et al., 1997; Prescott

& Koninck, 2002). Studying the physiological properties of these

neurons in lamina I, Prescott & Koninck, 2002) concluded that

‘repetitive’ (tonic in their classification scheme) and ‘phasic’ cells

respond in a graded fashion over a wide range of stimulus intensities

and their slow synaptic events and slow membrane time constant

promote temporal summation or integration of synaptic inputs.

‘Single’ neurons

‘Single’ neurons responded with only one, occasionally with two,

action potential at the onset of suprathreshold current steps. This may

indicate that these neurons are incapable of encoding stimulus inten-

sity through firing frequency but could follow high frequency stimula-

tion that may arise from various sources including burst firing or

rhythmically active neurons (Thomson et al., 1989). High frequency

stimulation of a neuron, however, can be accomplished only in those

cases when synaptic inputs generate synaptic potentials on various

areas of the somatodendritic membrane more or less at the same time.

Thus, ‘single’ neurons require a substantial coincident input from one

or different sources to allow the spatial summation of postsynaptic

potentials necessary for driving the neuron to fire. Once they are

activated they become incapable of generating any consecutive spikes

to subsequent incoming signals. To regain their responsiveness they

Table 2. Morphometric parameters of recorded and labelled neurons in laminae V–VII

Neuron type

Phasic(n¼ 10)

Repetitive(n¼ 15)

Slow(n¼ 8)

Single(n¼ 9)

Significance(Student’s t-test)

Average maximum cross-sectional area (mm2) 209� 19.69 268.55� 30.72 376.49� 37.61 209.32� 26.34 Phasic vs. slow, P< 0.01;repetitive vs. slow, P< 0.05;single vs. slow, P< 0.01.

Maximum diameter (mm2) 24.57� 2.63 27.42� 2.45 35.8� 3.91 21.26� 1.4 NSMinimum diameter (mm2) 13.38� 0.77 15.85� 1.09 17.47� 0.84 14.08� 0.79 NSAspect ratio 1.84� 0.16 1.81� 0.22 2.11� 0.3 1.51� 0.07 NS

NS, no significant differences.


542 P. Szucs et al.

need to be repolarized to resting membrane potential. Thus they can

also be regarded as onset detectors (Ruscheweyh & Sandkuhler, 2002).

In our sample most ‘single’ neurons were distributed in lamina

V–VI. Neurons with similar firing patterns were reported also in the

superficial and deep dorsal horn (Hochman et al., 1997; Prescott &

Koninck, 2002), where various types of primary afferents terminate

(Fyffe, 1984). These observations which show an overlapping between

the distribution of primary afferent terminals and ‘single’ neurons

Fig. 4 Dendritic branching patterns of neurons in laminae V–VII. (Column A) Averaged branching point distribution histograms of neurons in the four firing patterngroups. Number of dendritic branching points counted in 50 mm bins are plotted against the distance from the soma. (Columns B and C) Dendrograms constructedfrom three-dimensional reconstruction showing the length and branching pattern of dendrites originating from the cell body and Neurolucida reconstruction’s of arepresentative cell from each firing pattern group. Neurons in the ‘phasic’, ‘repetitive’ and ‘single’ groups had dendrites branching mainly within 100–150mmdistance from the soma. Cells in the ‘slow’ group presented dendrites that were continuously branching as they receded away from the cell body. Occasionally in the‘phasic’ and ‘repetitive’ groups a second distant accumulation of branching points occurred, which is represented on the histograms as a second small hump between300 and 600mm. Scale bars, 100mm. Numerical data in the histograms are presented as mean� SEM.



suggest, that ‘single’ neurons among many other possible sources may

receive innervation also from primary sensory neurons. This notion is

reinforced by the finding that Ia inhibitory interneurons, that are known

to be heavily innervated by primary afferents (Jankowska, 1992), have

also been classified as ‘single’-spiking neurons in the adult cat spinal

cord (Jankowska, 2001).

‘Slow’ neurons

‘Slow’ neurons showed unique physiological and morphological

characteristics that made them substantially different from the other

cell types. During suprathreshold depolarization, they fired a contin-

uous and low frequency train of action potentials without any obvious

sign of spike accommodation or attenuation. In addition, this was the

only cell type that showed ongoing spontaneous firing (not demon-

strated here) at resting membrane potential, when there was no

hyperpolarizing current injected. The spontaneous firing together with

the strong and elongated afterhyperpolarization that always followed

the action potentials resembled a pacemaker like activity. It also

appears to be important that all ‘slow’ neurons recorded in this study

were confined to the ventromedial grey matter of the spinal cord, where

Ole Kiehn and his collaborators observed rhythmically active neurons

in the neonatal rat spinal cord (personal communication). Ablation

studies carried out on embryonic and neonatal chicken and rats

unequivocally demonstrated that this area of the spinal grey matter

is essential in the generation of rhythmic motor behaviours like

locomotion (Ho & O’Donovan, 1993; Kjaerulff & Kiehn, 1996; Tresch

& Kiehn, 1999). Taking all of this together, we assume that ‘slow’

neurons may represent essential constituents of rhythm and/or pattern

generating spinal motor neural assemblies that are responsible for the

generation of flexor-extensor and left-right alternation. The findings

that the axons of some ‘slow’ neurons project to the ipsilateral motor

column, whereas others cross the midline and terminate in the con-

tralateral grey matter appear to reinforce this notion.

Acknowledgements

This was work supported by the Hungarian National Science Fund (OTKA T032075) and the Ministry of Welfare of Hungary (ETT 04–032/2000). Theauthors would also like to thank SUPERTECH (Hungary), for all their technicalsupport and Dr G. Veress for his help with the figure preparation.

Abbreviations

ACSF, artificial cerebrospinal fluid; AHP, afterhyperpolarization; DIC,differential interference contrast; ISI, interspike intervals.

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