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Developmental changes of transient potassium currents in large aspiny neurons in the neostriatum
Research report
Developmental changes of transient potassium currents in large aspiny
neurons in the neostriatum
Ping Deng, Zhiping Pang, Yuchun Zhang, Zao C. Xu*
Department of Anatomy and Cell Biology, Indiana University School of Medicine, 635 Barnhill Drive, MS 507, Indianapolis, IN 46202, USA
Accepted 5 August 2004
Available online 11 September 2004
Abstract
Developmental regulation of the potassium conductance is important for the maturation of neuronal excitability and the formation of
functional circuitry in the central nervous system (CNS). The rapidly inactivating A-type current is a major component of the voltage-
dependent outward potassium currents in the large aspiny (LA) neurons in the neostriatum. The large aspiny neurons play important roles in
the function of neostriatum in physiological and pathological conditions. Whole-cell patch-clamp recording was performed on acutely
dissociated neurons and brain slices to investigate the postnatal development of A-type current in the large aspiny neurons. The current
density of A-type current in large aspiny neurons was the highest at postnatal 13 days and gradually decreased during the development with
the lowest levels in adult animals. In comparison to postnatal 13 days, the steady-state inactivation curve shifted in depolarizing direction in
mature neurons. No significant changes in the voltage dependence of steady-state activation were observed during development. Consistent
with the decrease in the current density of A-type current during development, the latency to the first spike was dramatically shortened in
mature large aspiny neurons. These results suggest that the decrease of rapidly inactivating A-type potassium current during development
might contribute, at least in part, to the maturation of the membrane excitability of large aspiny neurons in the neostriatum.
D 2004 Elsevier B.V. All rights reserved.
Theme: Development and regeneration
Topic: Neurotransmitter system and channels
Keywords: Development; A-type potassium current; Membrane excitability; Whole-cell patch-clamp recording
1. Introduction
Large aspiny (LA) neurons are cholinergic interneurons
in the neostriatum
[3,12,48]
. Morphological studies have
demonstrated that LA neurons in rats have large somata
(2035 Am) with 35 extended primary dendrites bearing
few spines
[3,4]
. Compared to medium-sized spiny neurons,
LA neurons have relatively depolarized resting membrane
potentials (about
À60 mV) and show large-amplitude and
long-duration afterhyperpolarizations following action
potentials
[2,6,35,48]
. Moreover, LA neurons display a
hyperpolarization-activated cation current (I
h
) related sag in
membrane potential during intracellular negative current
injection
[21,23,48]
. These morphological and electrophy-
siological characteristics allow LA neurons to be easily
distinguished from other striatal neurons. Although they
only account for less than 2% of the neuronal population in
the neostriatum, LA neurons exert great influences on the
function of the basal ganglia by modulating the synaptic
transmission of spiny projection neurons and interneurons
[7,15,24,26]
. Maintaining the proper neuronal excitability is
essential for the function of LA neurons.
Voltage-dependent potassium currents are critical to the
maintenance of neuronal excitability through the regulation
0165-3806/$ - see front matter
D 2004 Elsevier B.V. All rights reserved.
doi:10.1016/j.devbrainres.2004.08.001
Abbreviations: ACSF, artificial cerebrospinal fluid; 4-AP, 4-amino-
pyradine; CNS, central nervous system; I
A
, rapidly inactivating A-type
potassium current; I
h
, hyperpolarization-activated cation current; I
kd
,
delayed rectifier potassium current; LA, large aspiny; TEA, tetraethylam-
monium; TTX, tetrodotoxin
* Corresponding author. Tel.: +1 317 274 0547; fax: +1 317 278 2040.
E-mail address: zxu@anatomy.iupui.edu (Z.C. Xu).
Developmental Brain Research 153 (2004) 97 107
www.elsevier.com/locate/devbrainres of resting membrane potential, the shaping of neuronal
firing patterns, the controlling of action potential frequency
and the determination of neuronal responses to synaptic
inputs
[16,37,45]
. Recent studies have shown that the
rapidly inactivating A-type potassium current (I
A
) is the
predominant component of depolarization-activated potas-
sium currents in LA neurons
[43]
. The A-type potassium
channels in LA neurons are attributable to the co-expression
of Kv4.2 and Kv4.1 subunits, and the Kv4.2 subunits are the
major constituents of the A-type potassium channels
[43,47]
. Another component of the outward currents,
namely the delayed rectifier potassium current (I
kd
), is
evident after inactivation of I
A
[43,47]
. During the postnatal
development of the central nervous system (CNS), potas-
sium conductance undergoes significant changes, which
indicates that it may play important roles in the maturation
of neuronal excitability as well as in the formation of
functional circuitry
[36]
. The developmental regulation of
potassium currents has been shown in many regions of the
CNS, such as the cerebral cortex, the hippocampus and the
cerebellar granule cells
[8,11,25,28,29,41,44]
. In the stria-
tum, the developmental changes of potassium currents in
spiny neurons have been elucidated
[46]
. In the present
study, we used whole-cell patch-clamp recording techniques
on acutely dissociated cells and brain slices to investigate
the development of I
A
in LA neurons. In brain slice
preparation, the neurons are relatively intact and close to
the physiological conditions. But the extended dendrites
compromise the voltage clamping of the cell and might
result in a space clamp error. In acutely dissociated neurons,
the space clamp problem is significantly reduced by
removing most of the dendrites. But the traumatic treat-
ments during the mechanical and enzymatic procedures
might have adverse effects on the physiological properties
of the cell. Therefore, both techniques were used in the
present study to validate the results.
2. Materials and methods
2.1. Brain slice and acute dissociation preparation
Male Wistar rats (Charles River, Wilmington, MA, USA)
were used in the present study. Experimental protocols were
institutionally approved in accordance with the National
Institutes of Health Guide for the Care and Use of
Laboratory Animals. All efforts were made to minimize
the suffering and the number of animals used.
Animals at the age of postnatal days 13, 79, 1416,
2227, 6090 (P13, P79, P1416, P2227, P6090) were
prepared for brain slices using procedures similar to those
previously described
[33]
. Briefly, the animals were
anesthetized with ketamineHCl (80 mg/kg, i.p.) and
decapitated. The brains were quickly removed and
immersed in ice-cold artificial cerebrospinal fluid (ACSF),
which was composed of the following (in mM): 130 NaCl, 3
KCl, 2 CaCl
2
, 2 MgCl
2
, 1.25 NaH
2
PO
4
, 26 NaHCO
3
and 10
glucose, pH 7.4, 295305 mOsm/l. Transverse striatal slices
of 280300 Am thickness were cut using a vibratome (VT
1000; Leica, Nussloch, Germany) and incubated in ACSF
for z1 h at room temperature (~24 8C) before being
transferred to the recording chamber. The slice was
submerged beneath the fluid surface and superfused con-
tinuously with oxygenated ACSF. The flow rate was
adjusted to 23 ml/min. Recordings were carried out at
room temperature.
Rats at the age of P13, P57, P1416 and P2527 were
used for acute dissociation using procedures similar to those
previously described
[49]
. In brief, rats were anesthetized
and decapitated. The brains were quickly removed, iced and
then blocked for slicing. The brain tissue containing
neostriatum was cut in 400 Am slices while bathed in a
low Ca
2+
, HEPES-buffered solution containing (in mM):
140 Na isethionate, 2 KCl, 4 MgCl
2
, 0.1 CaCl
2
, 23 glucose,
15 HEPES, pH=7.4, 300305 mosM/l. Slices were incu-
bated at room temperature in oxygenated ACSF. Then, the
slices were transferred into the low Ca
2+
buffer and regions
of striatum were dissected and placed in an oxygenated
HEPES-buffered Hanks balanced salt solution containing
13 mg/ml protease. After 2530 min of enzyme digestion
at 35 8C, tissue was rinsed three times in the low Ca
2+
buffer
and mechanically dissociated with a graded series of fire-
polished Pasteur pipettes. The cell suspension was plated
onto a 12 mm cover slip (Fisherbrand Coverglass; Fisher
Scientific, Pittsburgh, PA, USA), which was then placed in
the recording chamber.
2.2. Whole-cell patch-clamp recording
The same procedures were applied to the recordings on
brain slices and dissociated neurons. Recording electrodes
were prepared from borosilicate glass (Warner Instruments,
Hamden, CT, USA) using a horizontal electrode puller (P-
97; Sutter Instruments, Novato, CA, USA) to produce tip
openings of 12 Am (35 MV). Electrodes were filled with
an intracellular solution containing (in mM): 1