Centre de Recherche Fernand-Seguin and Department of Psychiatry, University of Montréal, Montreal, Quebec H1N 3V2, Canada
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ABSTRACT |
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Belleau, Marc L. and
Richard A. Warren.
Postnatal Development of Electrophysiological Properties of
Nucleus Accumbens Neurons.
J. Neurophysiol. 84: 2204-2216, 2000.
We have studied the postnatal development
of the physiological characteristics of nucleus accumbens (nAcb)
neurons in slices from postnatal day 1 (P1) to
P49 rats using the whole cell patch-clamp technique. The
majority of neurons (102/108) were physiologically identified as medium
spiny (MS) projection neurons, and only these were subjected to
detailed analysis. The remaining neurons displayed characteristics
suggesting that they were not MS neurons. Around the time of birth and
during the first postnatal weeks, the membrane and firing
characteristics of MS neurons were quite different from those observed
later. These characteristics changed rapidly during the first 3 postnatal weeks, at which point they began to resemble those found in
adults. Both whole cell membrane resistance and membrane time constant
decreased more than fourfold during the period studied. The resting
membrane potential (RMP) also changed significantly from an average of
50 mV around birth to less than
80 mV by the end of the third
postnatal week. During the first postnatal week, the current-voltage
relationship of all encountered MS neurons was linear over a wide range
of membrane potentials above and below RMP. Through the second
postnatal week, the proportion of neurons displaying inward
rectification in the hyperpolarized range increased steadily and after
P15, all recorded MS neurons displayed significant inward
rectification. At all ages, inward rectification was blocked by
extracellular cesium and tetra-ethyl ammonium and was not changed by
4-aminopyridine; this shows that inward rectification was mediated by
the same currents in young and mature MS neurons. MS neurons fired
single and repetitive
Na+/K+ action potentials as
early as P1. Spike threshold and amplitude remained constant
throughout development in contrast to spike duration, which decreased
significantly over the same period. Depolarizing current pulses from
rest showed that immature MS neurons fired action potentials more
easily than their older counterparts. Taken together, the results from
the present study suggest that young and adult nAcb MS neurons
integrate excitatory synaptic inputs differently because of differences
in their membrane and firing properties. These findings provide
important insights into signal processing within nAcb during this
critical period of development.
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INTRODUCTION |
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The nucleus accumbens (nAcb) is
an important telencephalic region that receives its most important
inputs from the prefrontal cortex, hippocampal formation, entorhinal
cortex, amygdala, and midline thalamic nuclei (Groenewegen et
al. 1980, 1982
, 1987
; Jayaraman 1985
; Kelley and Domesick 1982
;
Kelley and Stinus 1984
; Kelley et al.
1982
; Krayniak et al. 1981
; Newman and
Winans 1980
; Phillipson and Griffiths 1985
). The
primary output of the nAcb is to the ventral pallidum (Hakan et
al. 1992
; Yang and Mogenson 1985
; Zahm
and Heimer 1990
), which is known to be involved in the
activation of voluntary movements (Heimer et al. 1994
;
Swerdlow and Koob 1987
). The nAcb is believed to be a
center for the integration of limbic and motor systems (Mogenson
et al. 1980
). It appears to be involved in reinforcement
aspects of behavior (Cador et al. 1991
; Carlezon
and Wise 1996
; Joseph and Hodges 1990
;
Wise and Bozarth 1987
).
Many nAcb neurons receive glutamatergic inputs from diverse sources
(Finch 1996; O'Donnell and Grace 1995
)
that must be harmoniously integrated for proper output to be generated
by nAcb projecting neurons (O'Donnell and Grace 1995
).
Presumably, the anatomical substrate for this functional integration is
achieved during development through competition/cooperation
interactions between the different inputs to the nAcb following Hebbian
rules (Hebb 1949
).
The activity of single neurons largely depends on their synaptic inputs
and their membrane intrinsic properties. These two aspects of neuronal
organization are still immature at birth in several regions of the
neuraxis, and they develop interdependently during the postnatal
period. The influence of the environment during this period has been
recognized since the pioneering work of Wiesel and Hubel (Wiesel
and Hubel 1963, 1965
) in the visual system.
Today, it is widely believed that inappropriate neuronal activity
during a critical period will lead to permanent impairment of function
(e.g., Yuste and Sur 1999
). Whereas there is an
extensive body of literature on the developmental plasticity of sensory systems, comparable studies of limbic structures are few. Moreover, there is a growing body of evidence suggesting that diseases such as
schizophrenia may be the result of a disturbed development of limbic
structures (Falkai and Bogerts 1993
; Weinberger
and Lipska 1995
). Recently, an animal model using early
postnatal lesion of the subiculum, a limbic structure that has been
found to be abnormal in postmortem tissue from schizophrenic patients, mimics much of the symptomatology of schizophrenia excluding higher brain functions symptomatology (Flores et al. 1996a
;
Lipska et al. 1993
; Weinberger and Lipska
1995
). One area in which neurochemical changes have been
observed in that model is the nAcb (Flores et al.
1996a
).
It is probable that the functional maturation of the nAcb depends on a
fragile equilibrium between its different inputs, the disturbance of
which would probably lead to pathological states (Lipska et al.
1993, 1998
; Weinberger and Lipska
1995
). The aim of the present study was to describe the
postnatal development of the physiological properties of nAcb neurons
to understand their role in the integration of afferent inputs.
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METHODS |
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Rat pups 1-5 day old (P1-P5) were anesthetized by
hypothermia, and P6-P49 rats were anesthetized with
methoxyfluran vapor in a closed environment. The animals were then
quickly decapitated. The brain was taken out and submerged in
artificial cerebrospinal fluid (ACSF) at 4°C containing (in mM) 126 NaCl, 26 NaHCO3, 10 dextrose, 3 KCl, 1.3 MgSO4, and 1.25 NaH2PO4 with a pH of 7.4 when bubbled with a gas mixture of 95% O2-5%
CO2. Four hundred micrometer-thick slices
containing the nAcb were cut with the brain tilted 15° from the
parasagittal plane using a vibroslicer (Campden Instruments). Slices
were transferred to a submerged-type recording chamber and continually
superfused with ACSF at room temperature (20-22°C) at a rate of 1.5 ml/min. The nAcb was visualized under a stereomicroscope (Leica) using
the anterior commissure, the neostriatum, the septum, and the
ventricles as landmarks based on Paxinos and Watson
(1986). Slices were incubated for at least 1 h before recording.
Pipettes were pulled from thin wall borosilicate capillary glass with a
P-87 micropipette puller (Sutter Instrument). The pipettes had a
resistance between 5 and 13 M when filled with a solution containing
(in mM) 140 K+ gluconate, 2 MgCl2, 0.1 CaCl2, 1.1 EGTA,
10 HEPES, and 2 K2-ATP and the pH adjusted to
7.3 ± 0.05 with 8N KCl solution. In some instances, the internal
solution was diluted by 2-5% with distilled H2O
to improve seal formation. QX-314 (Alomone Laboratories; 1-2 mM) was
sometimes added to the recording pipette solution to block voltage-sensitive Na+ channels generating the
action potential. To study specific K+ channels,
the following agents were sometimes added to the superfusing ACSF:
tetraethyl ammonium (TEA; 25 mM) (see Nisenbaum and Wilson 1995
), 4-aminopyridine (4-AP; 100 µM or 2 mM), and CsOH or
CsCl (3 mM). When 25 mM of TEA was used, the concentration of NaCl was
equimolarly adjusted; no correction was made for the other agents.
The pipette was aimed at the nAcb under direct visual guidance. Whole
cell recording was achieved using the blind patch-clamp technique
(Blanton et al. 1989) with an Axoclamp-2B amplifier (Axon Instruments). The potential reference of the amplifier was adjusted to zero prior to breaking into the cell. The resting membrane
potential (RMP) was measured as soon as the whole cell configuration
was achieved, and the offset potential, measured on withdrawal from the
cell, was estimated by assuming that it drifted in a linear fashion
with time from the start of the recording session. A 10-mV junction
potential was subtracted from all membrane potential measurements
(Huguenard and Prince 1994
; Spigelman et al.
1992
).
The amplifier bridge balance was optimally adjusted. The ouput of the amplifier was fed to a LPF 200A DC amplifier/filter (Warner Instruments) and digitized at 0.5 to 10 kHz by a real-time acquisition system Digidata 1200 (Axon Instruments). Data acquisition and off-line analysis were done with the pClamp 6.0 software (Axon Instruments).
During current-voltage (I-V) curve recordings, two to four
sweeps at the same current level were routinely averaged on-line. Voltages were measured at the end of a current step episode of about
400 ms, and the input resistance (Rin)
was calculated in the linear range of the I-V curve around
the holding membrane potential. The membrane time constant
(m) was calculated by fitting a simple
exponential to an hyperpolarizing voltage response having amplitudes
between 3 and 8 mV to minimize the activation of voltage-gated ions
channels. Action potential characteristics including amplitude, duration, and threshold as well as spike train patterns, were examined
with supra-threshold depolarizing current pulses of varying duration
and amplitude.
To compare the physiological characteristics of nAcb neurons with
dorsal striatal neurons, the hyperpolarization sag and the afterhyperpolarization were measured as follows using the methods of
Kawaguchi (Kawaguchi 1992, 1993
). The
%sag was measured as 100 × (Vpeak
Vend)/(Vpeak
Vhold). The sag latency was the time difference between the onset of the current pulse and the time of
Vpeak. The amplitude and latency of
the spike afterhyperpolarization (AHP) were defined as the voltage and
time differences, respectively, from action potential threshold to the
dip voltage of the AHP. Results are presented as means ± SE.
Statistical analysis was performed using Sigmastat (SPSS) and
Statistica (Statsoft) softwares. When necessary, raw data were
logarithmically transformed to fulfill the requirements of parametric
statistical tests (Sokal and Rohlf 1995
).
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RESULTS |
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Whole cell recordings from 108 nAcb neurons were made in slices from P1-P49 rats. Because of the young age of some animals, it was easier to identify the core than the shell region of the nAcb and, therefore most neurons (91/108) were recorded in the former, whereas few (17/108) in the latter. However, we found no marked differences between the electrophysiological properties of core and shell neurons from animals of the same age groups, and data from both subnuclei were pooled in subsequent analyses.
Electrophysiological identification of cell types
Four types of neurons have been physiologically characterized in
the dorsal striatum (e.g., Kawaguchi et al. 1995), and
anatomical studies suggest that comparable classes of neurons are
present in the nAcb (Meredith et al. 1989
,
1992
; Pickel and Chan 1990
; Zahm
1992
). Medium spiny (MS) neurons form 90-95% of the neuronal population of the nAcb, and most of the neurons recorded in the present
study displayed the electrophysiological characteristics that have been
attributed to MS neurons (e.g., Kawaguchi 1997
). Indeed,
102 neurons (94%) were considered of the MS type because they
displayed a slowly depolarizing ramp at all developmental stages when
depolarized to or slightly above firing threshold with an intracellular
current injection (Fig. 1A,
arrow in middle panel). This is a characteristic of MS
neurons, but not of any other nAcb nor dorsal striatum interneurons
(O'Donnell and Grace 1993
, 1995
;
Pennartz and Kitai 1991
; Uchimura et al.
1989a
). In addition, most of these (n = 85;
83%) exhibited substantial instantaneous inward rectification when
hyperpolarized from RMP with intracellular current injection (Fig.
1A, arrowhead in left panel), another characteristic of MS neurons. In some MS neurons, a small depolarizing sag (usually <1%) was observed when large hyperpolarizing current pulses were injected (Fig. 1A, arrow in left
panel). Sags in MS neurons were never of comparable magnitude to
those found in large aspiny cholinergic (LA) neurons (Kawaguchi
1992
, 1997
; Kawaguchi et al.
1995
), and the characteristics of the sags did not change significantly with age. The remaining putative MS neurons
(n = 17; 17%) lacked inward rectification (e.g., Fig.
4A). They were all recorded in slices from animals younger
than P16, and, consequently, the absence of inward
rectification was considered a characteristic of immature MS neurons
(see Passive membrane properties).
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Of the remaining six neurons, three displayed a large and slow depolarizing sag in response to hyperpolarizing current pulses (Fig. 1B, arrow in left panel), a large amplitude and long duration AHP (Fig. 1B, arrow in middle panel), and a regular firing pattern (Fig. 1B, right). These characteristics distinguish them from MS neurons.
The other three neurons exhibited the characteristics of fast spiking (FS) neurons. All three neurons lacked significant amount of inward rectification (Fig. 1C, left), displayed comparatively short-duration action potential (Fig. 1C, middle) and high-frequency spike trains with little adaptation (Fig. 1C, arrow in right panel). One of these displayed an interrupted firing pattern in response to large depolarizing currents (Fig. 1C), whereas the other two exhibited a fast decaying spike train (not shown). Only the 102 putative MS neurons were retained for further analysis and developmental considerations.
Passive membrane properties
Passive membrane properties of nAcb MS neurons changed dramatically during the first few postnatal weeks (Fig. 2). In general, changes were more prominent during the first two postnatal weeks, and the rate of change tapered off during the third postnatal week.
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The RMP became more negative with age, averaging 59 ± 3 mV
(n = 9) during the first postnatal week and deepening
to
84 ± 1 mV (n = 34) after P21
(Fig. 2A). There was an even more dramatic change in
m, which decreased fourfold from an average of
143 ± 16 ms (n = 7) during the first postnatal
week to 33 ± 5 ms (n = 28) after P21
(Fig. 2B). The shortening of
m was
paralleled by a reduction of comparable magnitude in
Rin, which decreased from 2,091 ± 334 M
(n = 12) to 305 ± 40 M
(n = 34) between the first and fourth postnatal week
when measured in the nonrectified range around the RMP (Fig.
2C).
Characteristic I-V responses obtained from P7 and
P30 MS neurons are shown in Fig.
3. At P7, the I-V
relationship was very close to linearity up to 140 mV, more than 80 mV negative to the RMP (Fig. 3A). In contrast, at
P30, there was a striking inward rectification that is
detectable below
90 mV, only 10 mV negative to the RMP (Fig.
3B). The amount of rectification was estimated by
subtracting the Rin measured in the
hyperpolarized range (Fig. 2D) from the one measured around
the RMP (Fig. 2C). The results obtained are plotted as a
function of postnatal age in Fig. 2E. It is apparent that a
large proportion of neurons displayed no inward rectification before 2 wk of age, whereas all older neurons exhibited a significant amount of
rectification. Following an abrupt rise during the second postnatal
week, the absolute amount of rectification apparently decreased
afterward. However, the inward rectification appeared relatively
constant throughout the postnatal period studied when it was expressed
as a percentage of Rin measured in the
linear range (Fig. 2F). This suggests that the reduction in
inward rectification was related to the parallel decrease in
Rin in both linear and rectified
ranges (e.g., Fig. 2, C and D). This conclusion
is supported by the fact that there was a strong correlation between
inward rectification and Rin (r = 0.925, P < 0.001, n = 81).
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During the first postnatal week, the I-V relationship of 90% of neurons (9/10) was linear over an extended range of hyperpolarized voltage responses, showing that young MS neurons lack inward rectification (Figs. 3A and 4A). At the beginning of the second postnatal week, a large proportion of cells began to display an inward rectification in response to hyperpolarizing current pulses. Only 13% of neurons recorded (6/45) between P8 and P15 lacked inward rectification, whereas all neurons (n = 33) recorded from rats P16 and older displayed a significant amount of inward rectification in the hyperpolarized range of voltage responses. To see whether there was a relationship between RMP and the presence or absence of inward rectification, we compared the RMP of neurons of similar ages with and without inward rectification. Cells displaying inward rectification displayed significantly more negative RMP than neurons lacking inward rectification (Fig. 4B). Furthermore, a significant correlation was found between the RMP of rectifying neurons and age (r = 0.481, P = 0.027) but not between the RMP of nonrectifying neurons and age (r = 0.374, P = 0.170), suggesting that the appearance of inward rectification is concurrent with a deepening of the RMP.
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Discharge properties
The action potential of nAcb MS neurons of all ages was
characterized by a fast rising phase followed by a slower repolarizing phase (Fig. 5, A and
B). When depolarized with positive current pulse, the spike
threshold could be positively recognized as an inflection following a
slow depolarizing ramp (Fig. 5, A and B, arrows
in left panels). The slow depolarizing ramp preceding the action potential was the most consistent physiological characteristic of MS neurons; it was observed in all MS neurons regardless of age.
Different potassium, calcium, and/or persistent sodium conductances have been suggested as responsible for this slow depolarization (Kawaguchi et al. 1989; Kita et al.
1985a
,b
; Nisenbaum et al. 1994
).
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Some characteristics of the action potential including threshold and amplitude did not noticeably change with age (Fig. 6, A and B). In contrast, there was a striking shortening in the duration at half-amplitude of the action potential, especially during the first 12 postnatal days (Figs. 5, A and B, and 6C), but then it remained relatively stable from P14 until the end of the period studied (Fig. 6C). At all ages, the addition of the Na+ channel blocker QX-314 (1 or 2 mM) to the recording pipette internal solution caused a significant reduction in amplitude (from 54.7 ± 1.4 mV, n = 65 to 35.5 ± 3.4 mV, n = 15; ts = 5.674, P < 0.001, df = 78) and an increase in duration (from 2.5 ± 0.1 ms, n = 65 to 7.6 ± 1.4 ms, n = 15; ts = 6.149, P < 0.001, df = 78) of the action potential showing that voltage-dependent Na+ channels were involved in spike generation.
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In MS neurons of all ages, a relatively small AHP (9.9 ± 0.4 mV,
n = 70), similar to the one found in dorsal striatal MS
neurons (Kawaguchi 1997), followed the action potential.
There was no statistically significant change in AHP amplitude with age
(R2 = 0.05, P = 0.07, n = 70), but its latency decreased significantly (Fig.
6D), suggesting that the kinetics of the current underlying the AHP changed during postnatal development.
Nucleus accumbens MS neurons of all ages fired repetitively and usually
showed little to moderate adaptation when depolarized with DC injection
(Fig. 5, A and B, right). Their firing
frequency increased in a quasilinear fashion as a function of the
intensity of the injected depolarizing current. Younger neurons, with
their RMP closer to firing threshold (e.g., Fig. 2A) and
their larger input resistance (e.g., Fig. 2C), required less
depolarizing current to fire a first action potential than older
neurons. The increase in firing frequency as a function of the amount
of depolarizing current injected decreased with age for both the first
interspike interval of a train (Fig.
7A) and the average frequency
of the whole train (Fig. 7B). When the frequency of the
first interval of a train or of the whole train was plotted as a
function of injected current, the relationship was almost linear with
correlation coefficients all larger than 0.9. The slopes of these
linear fits were used to quantify the adaptation of MS neurons (Fig. 7,
C and D). Those from the spike train were
significantly smaller than the slope of the first interspike interval
(ts = 4.07, P < 0.001, df = 32), showing that there was a small but constant adaptation during a train. Adaptation was observed in all neurons, and
it did change significantly during postnatal development
(R2 = 0.082, P = 0.11, n = 32). Adaptation measured as a percent reduction in
frequency from the first interval averaged 6.5 ± 1.0%, showing
that adaptation was consistent but not very large.
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When depolarized from a holding membrane potential around 70 mV or
above, MS neurons from animals of all ages fired normal action
potentials (Fig. 8A,
left). In contrast, some neurons from young animals fired a
fast small amplitude action potential when depolarized from a holding
membrane potential around
90 mV (Fig. 8A,
right). In these neurons, sub-threshold depolarizing current pulses from a membrane potential of
90 mV consistently evoked a
depolarizing hump (Fig. 8A, arrow in right
panel), which was absent at
70 mV (Fig. 8A, left).
The shunting effect of this small depolarizing hump on the
Na+/K+ spike was most
striking during a spike train evoked from
90 mV. This started with a
small action potential followed by normal size action potentials (Fig.
8B, right) comparable to those evoked from
70 mV (Fig.
8B, left). The direct comparison of normal and shunted
action potentials showed that the rise time of the action potential was
little modified by the depolarizing hump, except for a modest but
consistent reduction in amplitude with no change in action potential
threshold (Fig. 8C). In contrast, the repolarization phase
of the action potential was much faster in presumably shunted than in
normal action potentials.
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The depolarizing hump could also be activated by membrane repolarization occurring at the end of a hyperpolarizing current pulse from RMP, and it evoked a single rebound action potential of normal size (Fig. 8D, arrow). However, with larger amplitude hyperpolarizations, the number of action potentials that could be evoked this way was limited to two, the second action potential being of small amplitude (Fig. 8D, inset). Overall, depolarizing humps were observed in 17 MS neurons, 15 of which were from animals less than 2 wk old, suggesting that their presence was transient during development.
Effects of potassium channels blockers
Previously described MS neurons in both nAcb and dorsal striatum
are characterized by a substantial inward rectification, whereas, in
the present study, putative MS neurons in slices from young animals
apparently lacked any detectable inward rectification. Inward
rectification in the hyperpolarized range is sensitive to certain
K+ channels blockers in nAcb MS neurons
(Uchimura et al. 1989a). To ascertain the absence of
inward rectification in young MS neurons and to study the nature
of the inward rectification expressed in juvenile MS neurons,
we have investigated the effects of specific K+
channels blockers including TEA, 4-AP, and Cs+.
Of these, Cs+ was found to be the most potent,
producing a voltage-dependent block of the inward rectification in
neurons in which measurable inward rectification was present (Fig.
9A). With long and/or with large hyperpolarizing pulses, the inward rectification often reversed to outward toward the end of the current pulse (Fig. 9A,
middle) and the Rin in the
hyperpolarized range became higher than that measured around the RMP
(e.g., Fig. 10, A and
B). Similar effects of Cs+ were
consistently observed in all tested neurons that displayed a
substantial inward rectification in the I-V relationship
(n = 10). In contrast, Cs+
produced no detectable effects in neurons in which there was no
evidence of an inward rectification (n = 5). The
effects of Cs+ on inward rectification are summarized in
Fig. 10. The Rin of young neurons
lacking inward rectification (Fig. 10A,
) was not changed
and remained linear with Cs+ added to the bath
(Fig. 10B,
). In contrast, the inward rectification of
older neurons (Fig. 10A,
) reversed to outward when
Cs+ was added to the perfusing medium (Fig.
10B,
). An analysis of covariance showed that the effects
of Cs+ were statistically significant
(Fs = 10.12, P = 0.004, df = 28).
|
|
TEA also decreased the inward rectification in MS neurons
displaying inward rectification (n = 3), but in a
voltage-independent manner. No inward to outward reversals in the
rectification at more hyperpolarized potentials were observed (Fig.
9B; note that the action potentials are truncated because
each trace represent the average of 2 sweeps, see Fig.
11B). In two neurons lacking inward rectification, TEA produced no detectable effect in their response to hyperpolarizing current pulses, although it had marked effects on their spiking characteristics (not shown). In contrast to
Cs+ and TEA, 4-AP had no statistically significant effects
on the response of MS neurons to hyperpolarizing current pulses
(ts = 0.52, P = 0.613, n = 9) despite its striking effects on the firing characteristics of all
neurons tested (Fig. 9C; note that the action potentials are
truncated because each trace represent the average of 2 sweeps). It was
also tested on three neurons which lacked inward rectification and
produced no significant effects on their I-V relationships.
|
The effects of K+ channel blockers on the
firing characteristics of MS neurons were also examined. Regardless of
age, Cs+ produced no significant effects on any
characteristics of the action potential including threshold
(ts = 1.2, P = 0.24, df = 25), peak (ts = 0.36, P = 0.72, df = 28) duration at half-amplitude (ts = 0.45, P = 0.66, df = 24; Fig. 11A). In contrast, TEA produced marked
effects on spike trains. These effects were qualitatively similar to
those described by Kita et al. (1985b) in dorsal
striatum MS neurons. Following an initial fast action potential (Fig.
11B, arrowhead), the remaining spike train was replaced by a
long depolarized plateau that could last several hundred milliseconds
when large depolarizing current pulses were used (Fig. 11B,
arrow). 4-AP also increased the duration of the action potential but
produced less dramatic effects than TEA (e.g., Fig. 9C,
middle). Before producing its maximal effects on the action
potential, 4-AP reduced the latency to the first spike discharge (Fig.
11C) and substantially reduced the amplitude of the AHP
(Fig. 11C, arrow) with no apparent effect on the action
potential threshold (ts = 1.11, P = 0.29, df = 16). All the effects of
K+ channels blockers on the membrane and
firing characteristics were fully reversible.
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DISCUSSION |
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Nucleus accumbens MS neurons were recorded in slices during
postnatal development from the day after birth (P1) until
early adulthood (P49). During that period, the basic
membrane and firing properties of MS neurons changed dramatically. Soon
after birth, they displayed some basic properties of neurons such as
negative RMP and the capacity to fire fast
Na+/K+ action potentials.
However, several aspects of the membrane and firing properties of young
MS neurons were remarkably different from those found in mature MS
neurons. During the postnatal period studied, the RMP became more
negative and the Rin and decreased severalfold. In addition, young MS neurons fired
Na+/K+ action potentials
more readily than older ones and lacked inward rectification during the
first postnatal week. By P49, the characteristics of MS
neurons were not noticeably different from those reported for adult
animals. However, our results showed that immature MS neurons do not
display membrane potential bistability, which suggests that
input/output integration is different in mature and immature nAcb.
Nature of immature nAcb neurons
MS neurons are the only known projection neurons in the nAcb, and
they are also the most abundant, forming 90-95% of the population. Despite the young age of some of our preparations, the large majority (94%) of recorded neurons were positively identified as MS neurons since they displayed at least one electrophysiological property of MS
neurons that is not found in other nAcb neuronal types. In nAcb and
dorsal striatum, only MS neurons display a depolarizing plateau
preceding the action potential when depolarized from RMP, and a
powerful instantaneous inward rectification sensitive to Cs+ when hyperpolarized from RMP (Chang
and Kitai 1986; Kawaguchi 1992
,
1993
, 1997
; Kawaguchi et al.
1989
, 1990
, 1995
;
Lopes da Silva et al. 1984
; O'Donnell and Grace
1993
; Pennartz et al. 1991
; Uchimura et
al. 1989a
,b
; Yang and Mogenson 1984
; Yim
and Mogenson 1982
, 1988
). In the present study,
all putative MS neurons in preparations older than P15, and
72% of putative MS neurons in preparations from P1 to
P15 animals displayed both characteristics. A subset of
neurons from animals P15 and younger lacked inward rectification, but they showed a slow ramp depolarization preceding the
action potential and, for this reason, were regarded as immature MS
neurons. The absence of inward rectification has also been reported in
subsets of MS neurons from kitten caudate nucleus (Cepeda et al.
1991
) and rat neostriatum (Tepper et al. 1998
), supporting the idea that the lack of inward rectification is a characteristic of immature MS neurons.
Six other neurons were classified as non-MS because they had neither of
the two physiological characteristics of MS neuron and displayed
properties suggesting that they belonged to other neuronal classes
types. Indeed, neurons with large depolarizing sag and important AHP
have been morphologically identified as large aspiny cholinergic
neurons in the dorsal striatum (Kawaguchi 1993;
Kawaguchi et al. 1995
), suggesting that cholinergic
interneurons have similar physiological properties in the nAcb. The
neurons we have named fast spiking neurons have similar physiological characteristics to a class of GABAergic interneurons also found in the
dorsal striatum (Kawaguchi et al. 1995
).
Development of membrane and firing properties of MS neurons
All putative MS neurons recorded prior to P7 lacked
inward rectification. Afterward, the proportion of rectifying neurons recorded increased constantly with aging so that all MS neurons displayed inward rectification after P15. This is in
contrast with an in vivo study in rat dorsal striatum in which the
proportion of MS neurons expressing inward rectification also increased
with age but was only 40% in P30-P42 animals and 80% in
adults (Tepper and Trent 1993; Tepper et al.
1998
). These discrepancies might reflect functional differences
between dorsal striatum and nAcb neurons or might be due to differences
in recording techniques.
In absolute terms, inward rectification was larger in cells from younger animals and decreased with age (e.g., Fig. 2E) but, since this decrease was parallelled by a decrease in Rin of comparable magnitude, it appeared as a somewhat all or none phenomenon readily integrated with other membrane properties and was not a progressive change in membrane properties such as the change in RMP. In contrast, the progressive increase in the proportion of neurons displaying inward rectification during the first two postnatal weeks suggests that the regulation of the expression of this phenotype is age dependent.
Developmental changes in basic membrane properties included a large
decrease in Rin paralleled by a
shortening in . Similar findings have been described in other
structures throughout the neuraxis (McCormick and Prince
1987
; Pirchio et al. 1997
; Ramoa and
McCormick 1994
; Spigelman et al. 1992
;
Tepper and Trent 1993
; Tepper et al.
1998
; Warren and Jones 1997
; Zhou and
Hablitz 1996
). The concomitant decrease in
Rin and
m
with age implies an increase in ion channel density as previously noted
(McCormick and Prince 1987
; Spigelman et al.
1992
; Zhou and Hablitz 1996
), assuming that the
specific membrane capacitance does not change with age (Deuchars
and Thomson 1995
, 1996
). Membrane resistance
also depends on the surface area of the cell (e.g., Warren and
Jones 1997
), but we do not have any morphological data to
confirm that MS neurons actually grow during the period covered by the
present study. In the dorsal striatum, although it was found that MS
neurons soma do not grow between P6 and adulthood, there is
a large increase in the number of dendritic spines and a thickening of
the dendrites (Tepper and Trent 1993
; Tepper et
al. 1998
), suggesting that changes in
Rin and
are parallelled by an
increase in cell membrane surface. The higher
Rin and thus a smaller electrotonic
length in newborns may help MS neurons to integrate synaptic inputs in
a more efficient manner; this could compensate for their lack of
organization (McCormick and Prince 1987
;
Spigelman et al. 1992
). Our
Rin and
values are much larger
than those reported by others for MS neurons (Kawaguchi et al.
1989
; Kita et al. 1984
; O'Donnell and
Grace 1993
). Whereas part of the differences can be attributed
to the young age of some of our preparations, much of the differences
can be explained by the fact that we used the whole cell recording
technique, which consistently yielded much higher values for these
parameters than sharp electrode recordings. However, the fact that the
experiments were conducted at room temperature may have somewhat
contributed the long
by slowing membrane conductances.
Several possibilities have been advanced to explain why the RMP is more
depolarized in immature neurons: these include a lower K+ conductance (Spigelman et al.
1992), an inactive
Na+/K+/Cl
co-transport extrusion mechanism (Zhang et al. 1991
),
and an immature Na+/K+
ATPase (Fukuda and Prince 1992
). In the present study,
we found that neurons expressing inward rectification displayed lower
membrane potential than neurons of the same age without this
characteristic, suggesting that a portion of the hyperpolarization of
the RMP is related to or co-expressed with inward rectification.
Short-duration action potentials were present as early as
P1. With low concentrations of QX-314 in the patch pipette,
the action potentials were much wider and of lower amplitude showing that functional voltage-dependant Na+ channels
are already involved in the depolarizing phase of the action potential
at birth. The duration of the action potential decreased by more than
half during the period studied, whereas its amplitude and threshold did
not significantly change. The average amplitude and threshold measured
in the present study are in the range of those reported in nAcb of
adult rats (O'Donnell and Grace 1993), supporting our
finding that these parameters do not significantly change during
postnatal development. In rat dorsal striatum in vivo, the action
potential threshold was also found to be constant during postnatal
development, although its amplitude was smaller between P6
and P10 than at later ages, but it did not change
significantly after that (Sharpe and Tepper 1999
).
Developmental changes in action potential duration and amplitude have
also been found in kitten caudate nucleus (Cepeda et al.
1991
). A maturation of sodium channels or an increase in their
density might explain why the action potential is shorter in older
animals. A parallel maturation of the K+ delayed
rectifier (KDR) and the fast sodium currents on
which the action potential threshold depends (Johnston and Wu
1995
) could explain the stability of the threshold during
postnatal development.
MS neurons fired both single action potentials and trains of spikes,
the frequency of which rose with the amount of injected current at all
ages, in agreement with findings in other brain structures
(McCormick and Prince 1987; Ramoa and McCormick
1994
; Warren and Jones 1997
; White and
Sur 1992
; Zhang et al. 1991
). With aging, more
depolarizing current was needed to generate action potentials in MS
neurons. Thalamic neurons are similarly affected by age (Ramoa
and McCormick 1994
; Warren and Jones 1997
),
whereas, in contrast, the current threshold of cortical pyramidal
neurons decreased with age (McCormick and Prince 1987
).
In the present study, we have identified two factors that could
contribute to this phenomenon. First, the RMP became more negative with
age, while the spiking threshold remained constant throughout
development, meaning that, even with a constant membrane
Rin, larger currents would be needed
to bring the membrane to spiking threshold and to keep it there during
sustained activity because of the increased ionic repolarizing strength
between spikes. Second, Rin decreased significantly with age, so that more current was needed to reach spiking threshold and this even if the RMP had remained constant. In
addition, the significant decrease in AHP latency with age suggests
that the amount of current needed to increase spiking frequency during
a train had to counteract larger Ca2+-dependent
K+ conductances with age.
In a subset of neurons we have observed a depolarizing hump that was
activated when the membrane was depolarized from 90 mV but not
70
mV. When activated on release from an hyperpolarizing current pulse, it
was sufficient to generate one or two rebound action potential. These
characteristics are reminiscent of low-threshold Ca2+ spike (LTS), which has been extensively
studied in thalamic neurons and has also been described in a subset of
nAcb neurons (O'Donnell and Grace 1993
). With
sufficient depolarization, the hump was overridden by a somewhat
shunted action potential. In contrast, the first rebound spike at the
end of a hyperpolarizing current pulse appeared normal and was followed
by a depolarizing hump. With larger hyperpolarization, a second,
smaller action potential was evoked. One plausible explanation is that
the depolarizing hump increased some ionic conductances, possibly
Ca2+-dependent K+ current
if the depolarizing hump is actually a LTS, that shunt a normal action
potential. Nevertheless, the depolarizing humps were of small amplitude
and could be compared with LTS found in thalamic relay neurons during
early postnatal development (e.g., Warren and Jones
1997
). On the other hand, we have not positively identified
these as LTS with the use of specific Ca2+
blockers, and, therefore we cannot exclude other possibilities such as
the involvement of electrotonic coupling or of dendritic or initial
segment spike (Grace 1990
).
Overall, changes in membrane and firing properties of nAcb MS neurons
occurred mostly during the first 3 postnatal weeks, whereas later,
changes could not be discriminated from interneuronal variability.
Firing characteristics appeared to reach adult values around
P15, whereas membrane properties including the RMP,
Rin, and appear to mature about 1 wk later. This is in close agreement with similar studies performed in
the dorsal neostriatum (Misgeld et al. 1986
;
Tepper and Trent 1993
; Tepper et al.
1998
), suggesting that the nAcb and dorsal striatum mature
during overlapping postnatal period. As remarked above, the only
notable difference is the presence of inward rectification in all nAcb
MS neurons after P15, and the implications for this
difference are discussed below.
Functional perspective
In vivo experiments have shown that MS neurons exhibit membrane
potential bistability and that this characteristic has a profound impact on the signal transfer characteristics of these neurons (Gerfen and Wilson 1996; Kincaid et al.
1998
; Nisenbaum and Wilson 1995
;
Nisenbaum et al. 1994
; O'Donnell and Grace
1995
; Wilson 1993
; Wilson and Kawaguchi
1996
; Xu et al. 1991
). Nucleus accumbens MS
neurons recorded in vivo in adult animals show a pattern of spontaneous
activity consisting of brief episodes of firing separated by long
periods of silence (O'Donnell and Grace 1993
,
1995
). Intracellular recordings of MS neurons in vivo in
both nAcb (Finch 1996
; O'Donnell and Grace
1995
; Yim and Mogenson 1988
) and dorsal striatum
(Buchwald et al. 1973
; Calabresi et al.
1990
; Finch 1996
; Hull et al.
1970
; Wilson and Groves 1981
; Wilson and
Kawaguchi 1996
; Yim and Mogenson 1988
) have
shown that active and silent episodes correspond to two different
stable membrane potential states about 20 mV apart: an hyperpolarized
silent state around
80 mV and a depolarized active state around
60
mV. MS neurons fire only when in the depolarized state with the spikes
often occurring in bursts. Membrane potential bistability in MS neurons
appears to be produced by the interplay between afferent excitatory
synaptic inputs and intrinsic membrane K+
conductances (Wilson and Kawaguchi 1996
). Following
lesion or reversible inactivation of hippocampal excitatory inputs in
vivo, nAcb MS neurons remain in the hyperpolarized state, suggesting that the depolarized state requires hippocampal inputs
(O'Donnell and Grace 1995
). In slices maintained in
vitro, the membrane potential of MS neurons remains around
80 to
90
mV, corresponding to that of the in vivo hyperpolarized state
(Chang and Kitai 1986
; O'Donnell and Grace
1994
; Uchimura et al. 1989b
). No depolarized
episodes are seen presumably because active excitatory synaptic inputs to the nAcb are cutoff (O'Donnell and Grace 1995
;
Wilson and Kawaguchi 1996
).
A low RMP such as that found in vitro in mature MS neurons is an
essential condition for the appearance of membrane potential bistability in MS neurons. Indeed, a low RMP coupled with the possibility of a stable plateau depolarization of 20 mV will maintain the membrane potential close to spiking threshold so that weak excitatory inputs from other sources will make the cell fire (e.g., O'Donnell and Grace 1995). The membrane properties in
young MS neurons are quite different from those present in mature
animals, suggesting that membrane potential bistability is unlikely to be the primary mode of input integration in MS neurons during the first
postnatal weeks. We have found that MS neurons RMP did not reach a
value comparable to adults (around
80 mV) before the end of the third
postnatal week, suggesting that membrane bistability could not be fully
developed before that time and that until then MS neurons will respond
differently to converging excitatory inputs. Indeed, membrane potential
bistability is not consistently observed in dorsal striatum MS neurons
in anesthetized rats before the end of the fourth postnatal week when
the proportion of MS neurons expressing inward rectification has
reached adult levels (Sharpe and Tepper 1999
;
Tepper et al. 1998
). In the nAcb, we have found that
inward rectification is expressed in all MS neurons by P16
and later, raising the possibility that bistability appears earlier in
the nAcb than in dorsal striatum. In addition, if the expression of
inward rectification is a requirement to membrane potential
bistability, than it is potentially expressed in all nAcb MS neurons
but only in a subpopulation of MS neurons in the dorsal striatum.
It has been suggested that inward rectification plays an important role
in membrane potential bistability (Wilson and Kawaguchi 1996). Its absence in most cells during the first postnatal
week indicates that MS neurons will not display membrane potential bistability during early postnatal life. In MS and some other types of
neurons, inward rectification is produced by a voltage-gated K+ current similar to
IKir (Nisenbaum and Wilson
1995
). This current may also be involved in the maintenance and
stability of relatively negative passive RMP (Nichols and
Lopatin 1998
).
The functional differences between young and mature MS neurons could be
important throughout a period during which activity-dependent development and stabilization of synaptic inputs is probably occurring in the nAcb. The nAcb receives putative excitatory glutamatergic inputs
from various sources that are not fully developed at birth, so the nAcb
is likely to complete its development in parallel with those
structures. Our results suggest that young MS neurons require smaller
excitatory synaptic inputs to be activated because of their more
depolarized RMP and their higher Rin.
This should lead to more frequent synaptically driven firing despite
the fact that MS neurons in younger animals are likely to receive
weaker synaptic input than in adults because the areas projecting to the nAcb are themselves immature. On the other hand, these synaptic inputs are probably endowed with greater plasticity at birth, since
during the first postnatal week at their relatively depolarized membrane potential, N-methyl-D-aspartate (NMDA)
receptors will be readily activated favoring
Ca2+-dependent plasticity. An interesting and
challenging question is as follows: what triggers the expression of
inward rectification in MS neurons? One possibility is that inward
rectification is expressed in MS neurons in an activity-dependent
manner following the arrival of a glutamatergic innervation
(Moody 2000). Indeed, early postnatal disruption of
hippocampal and cortical innervation of the nAcb results in
long-lasting neuromolecular changes in the nAcb accompanied by an
increase in behavioral sensibility to psychostimulants (Flores
et al. 1996a
,b
; Lipska et al. 1998
). This could
be related to changes in K+ conductances
(Premkumar and Ahern 1995
; Rosenzweig-Lipson
et al. 1997
; Wang and Grahame-Smith 1992
). An
electrophysiological characterization of MS neurons of different ages
that were deafferented at birth may help to answer this question.
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ACKNOWLEDGMENTS |
---|
We are grateful to Drs. Massimo Avoli, Arlette Kolta, and James P. Lund for commenting on earlier versions of the manuscript.
This work was supported by the Fonds de la Recherche en Santé du Québec and by the National Science and Engineering Research Council of Canada. R. A. Warren was supported by a fellowship from the Fonds de la Recherche en Santé Mentale du Québec.
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FOOTNOTES |
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Address for reprint requests: R. A. Warren, Centre de Recherche Fernand-Seguin, 7331 Hochelaga St., Montreal, Quebec H1N 3V2, Canada (E-mail: richard.warren{at}umontreal.ca).
Received 22 March 2000; accepted in final form 19 June 2000.
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REFERENCES |
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