Department of Physiology and Neurobiology, University of Connecticut, Storrs, Connecticut 06269
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ABSTRACT |
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Gabel, Lisa A. and
Joseph J. LoTurco.
Electrophysiological and Morphological Characterization of
Neurons Within Neocortical Ectopias.
J. Neurophysiol. 85: 495-505, 2001.
Focal developmental abnormalities
in neocortex, including ectopic collections of neurons in layer I
(ectopias), have been associated with behavioral and neurological
deficits. In this study, we used infrared differential interference
contrast microscopy and whole cell patch-clamp to complete the first
characterization of neurons within and surrounding neocortical
ectopias. Current-clamp recordings revealed that neurons within
ectopias display multiple types of action potential firing patterns,
and biocytin labeling indicated that ~20% of the cells in
neocortical ectopias can be classified as nonpyramidal cells and the
rest as atypically oriented pyramidal cells. All cells had spontaneous
excitatory (glutamatergic) and inhibitory (GABAergic) postsynaptic
currents. Exhibitory postsynaptic currents consisted of both
N-methyl-D-aspartate (NMDA) receptor-mediated and AMPA/kainate (A/K) receptor-mediated currents. The NMDA
receptor-mediated component had decay time constants of 15.35 ± 2.2 (SE) ms, while the A/K component had faster decay kinetics
of 7.6 ± 1.7 ms at 20 mV. GABAA
receptor-mediated synaptic currents in ectopic cells reversed at
potentials near the Cl
equilibrium potential
and had decay kinetics of 16.65 ± 1.3 ms at 0 mV. Furthermore we
show that cells within ectopias receive direct excitatory and
inhibitory input from adjacent normatopic cortex and can display a form
of epileptiform activity.
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INTRODUCTION |
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Focal neocortical
malformations, including focal cortical dysplasias, microgyrias,
nodular heterotopias, and molecular layer ectopias, have been
associated with several neurological disorders (Arnold et al.
1991; Battaglia et al. 1997
; Galaburda et
al. 1985
; Hannan et al. 1999
; Humphreys
et al. 1990
; Lombroso 2000
; Palmini et
al. 1991a
,b
; Trottier et al. 1994
). To better
understand the role these malformations play in neurological
malfunction, studies must be directed at understanding the cellular
physiology of cells within and surrounding these malformations. In the
past several years, animal models of several types of focal cortical
malformations have been developed, and these models display pathologies
similar to the human conditions. For example, microgyria and double
cortex in rats have been shown to be associated with epileptiform
activity in vitro and in vivo (Chen et al. 2000
;
Jacobs et al. 1996
, 1999
; Lee et al.
1997
; Luhmann et al. 1998
; Prince and
Jacobs 1998
; Prince et al. 1997
).
Ectopias, clusters of misplaced cells in layer I of the neocortex, have
been associated in humans with developmental dyslexia (Galaburda
et al. 1985) and psychomotor retardation (Caviness et
al. 1978
). In brains of dyslexics, as many as 30-140
individual ectopias measuring 700-1,000 µm in diameter, which often
extend into and disrupt the laminar pattern in cortical layers 2/3,
were identified (Galaburda et al. 1985
; Kaufmann
and Galaburda 1989
). The formation of ectopias appears to occur
as a result of disrupted migration caused by either abnormal
interactions between migrating neuroblasts and radial glial fibers
(Caviness et al. 1978
) and/or disruptions in the pia and
layer 1 (Caviness et al. 1978
; McBride and Kemper
1982
).
Ectopias virtually identical to those described in humans have been
found in three strains of autoimmune mice: NZB/BlNJ, BXSB/MPJ, and
NXSM-D/Ei. Ectopias in these animals typically contain more than 50 cells, are located in either somatosensory or frontal/motor cortices,
and are present in the brains of 40-85% of mice (Boehm et al.
1996; Denenberg et al. 1991
; Sherman et
al. 1990
, 1994
). Although the majority of mice with ectopias
have only one ectopia, a small percentage have multiple ectopias
(Boehm et al. 1996
; Denenberg et al.
1991
; Sherman et al. 1990
). Behaviorally, mice with neocortical ectopias are impaired in certain learning tasks, such
as spatial and nonspatial working memory (Balogh et al.
1998
; Boehm et al. 1996
; Denenberg et al.
1991
; Schrott et al. 1993
; Spencer et al.
1986
), and in processing rapid auditory stimuli (Clark
et al. 2000
; Frenkel et al. 2000
).
It remains unclear how ectopias are mechanistically linked to
deficits in either humans or animal models. While altered local cortical and cortico-thalamic connections have been associated with
ectopias (Jenner et al. 2000), the cellular physiology
of cells within ectopias has yet to be described. In this study we have
used whole cell patch-clamp techniques to characterize the physiological properties of cells within neocortical ectopias in
NZB/BlNJ and NXSM-D/Ei mice. This paper provides evidence that layer I
ectopias contain neurons with diverse cellular physiologies and
morphologies, contain both GABAergic and glutamatergic synapses, receive direct inhibitory and excitatory connections from surrounding normatopic cortex, and, in some cases, can display epileptiform activity.
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METHODS |
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Whole cell recordings were made from neurons within and
surrounding layer I neocortical ectopias. For the purposes of this study, an ectopia is defined as a large, cluster of misplaced cells in
layer I of the neocortex. New Zealand black (NZB/BlNJ) and the
recombinant in-bred NXSM-D/Ei mice, derived from a cross between
NZB/BlNJ and SM/J mouse strains (Eicher and Lee 1990), both spontaneously develop large single-layer I ectopias located primarily in somatosensory cortices (Denenberg et al.
1991
; Sherman et al. 1987). Based on the
similarity in both location and size of the ectopias, the data have
been collapsed across these two strains of mice.
Preparation of brain slices
Adult male and female NXSM-D/Ei and NZB/BlNJ mice (30-388 days
postnatal) were deeply anesthetized with halothane and decapitated. Following decapitation, the brains were removed rapidly from the skull
and immersed in an ice-cold, oxygenated sucrose-artificial cerebrospinal fluid (aCSF) solution containing (in mM) 124.0 sucrose, 5.0 KCl, 2.0 MgCl2, 1.23 NaH2PO4
(2 · H2O), 23.8 NaHCO3,
and 2.0 CaCl2; pH = 7.4. The brains were
blocked, and 300-µm-thick coronal serial sections were cut using a
Vibroslice (Campden Instruments, London, UK). Slices were transferred
to a petri dish containing ice-cold, oxygenated aCSF and examined with
oblique illumination under a dissecting microscope to identify slices
containing ectopias (Fig. 1, A
and B). aCSF contained (in mM) 124.0 NaCl, 5.0 KCl, 2.0 MgCl2, 1.23 NaH2PO4
(2 · H2O), 23.8 NaHCO3,
and 2.0 CaCl2; pH = 7.2, osmolarity = 300 ± 5 mOsm/l. Slices selected for recording were affixed to
18-mm circular microscope cover slips (Fisher Scientific, Pittsburgh,
PA) with plasma/thrombin clots. Slices were maintained at room
temperature (20-22°C) in aCSF for 1 h in an oxygenated holding
chamber before recording began. Prior to whole cell recording, the
18-mm circular microscope cover slips containing slices were mounted
onto the stage of a Nikon Eclipse 400 microscope. Recordings were
performed at either room temperature (20-22°C) or 34-35°C.
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Whole cell recordings
Whole cell recordings were made from cells within neocortical
ectopias in somatosensory cortex. Slices were visualized, and neurons
within ectopias were targeted using infrared differential interference
contrast (IR-DIC) microscopy. Electrodes were pulled from capillary
tubing (Garner Glass N51A, Garner Glass, Claremont, CA) using a
Narishige multi-step electrode puller (Model PP-830) and had
resistances of ~10 M. The intracellular solution, used for
voltage-clamp recordings, contained (in mM) 120.0 cesium gluconate, 10.0 ethylene glycol-bis (
-aminoethyl ether)-N,
N,N',N'-tetraacetic acid, 10.0 HEPES, 1.0 CsCl, and 1.0 MgCl2; the pH was adjusted to 7.4 ± 0.05 with HCl 5.0 N and osmolarity was adjusted to 270 ± 5 mOsm/l. The
intracellular solution used for current-clamp recordings contained (in
mM) 120.0 potassium gluconate, 10.0 HEPES, 1.0 KCl, and 1.0 MgCl2; the pH was adjusted to 7.4 ± 0.05 with HCl 5.0 N and osmolarity was adjusted to 270 ± 5 mOsm/l. In
some experiments, pharmacological agents were used to selectively
isolate the individual synaptic responses. Picrotoxin (PTX) and
bicuculline methiodide (BMI) were purchased from Sigma Chemicals (St.
Louis, MO). d-(
)-2-amino-5-phosphonovaleric acid (d-AP5) and
6,7-dinitroquinoxaline-2,3-dione (DNQX) were purchased from Tocris
Cookson (St. Louis, MO).
Voltage- and current-clamp recordings were made using an Axon Instruments 200B amplifier (Axon Instruments, Foster City, CA) and low-pass filtered at 1 kHz. Currents were digitally sampled at 10 kHz and monitored with pCLAMP 7.0 software (Axon Instruments) running on a PC pentium computer.
A bipolar stimulating electrode was placed in sites adjacent and below
ectopias (~400 µm), using pulses of 100 µs and 50-400 µA,
unless otherwise noted. To examine the synaptic properties of ectopias,
both paired-pulse and potentiation protocols were used. Maximal
stimulation was determined by applying increasing stimulation using
pulses of 100 µs and between 50 µA to 1 mA at 15-s intervals.
Paired pulses, at 50-75% maximal stimulation, were recorded at
inter-stimulus intervals of 50, 100, 200, 300, 400, 500, 600, 700, and
800 ms. Potentiation was induced using 7 cycles of a 50 Hz stimuli (80 ms) applied at 0.1 Hz while the cells were depolarized to about 20
mV, after a baseline level of activity was recorded for ~10 min.
Potentiation was defined as a
20% increase in postsynaptic potential
(PSP) amplitude immediately following tetanization, which was sustained
for
10 min.
Data analysis
Spontaneous and evoked postsynaptic currents (PSCs) were detected and measured using Mini Analysis 5.0 software (Synaptosoft), which identifies spontaneous currents on the basis of several criteria, including threshold, amplitude, decay, and rise time properties of each event. Although the program is designed to automatically detect events based on these criteria, each event was manually selected based on rise time and decay properties. In some cases, PSPs were identified and analyzed using Clampfit 8 software (Axon Instruments). Kinetic analysis of the PSCs were performed by fitting the decaying phase of the PSCs with a single-exponential function. To increase the signal to noise ratio of traces that had peak amplitudes that were just above noise, the traces were filtered at 1 kHz and then analyzed based on rise-time and decay properties. All data throughout this report are expressed as means ± SE unless indicated otherwise.
Histological procedures
In all recordings, biocytin (Sigma), ~1-2%, was included in
the internal pipette solution. Following electrophysiological recording, the tissue was fixed in 4% paraformaldehyde for 24 h at
4°C. The tissue was rinsed in phosphate buffered saline (PBS) prior
to histochemical analysis. Sections (300 µm) were treated in 0.5%
H2O2 in PBS for 20-30 min
at room temperature (RT) to eliminate endogenous peroxidase activity.
Sections were then permeabilized and blocked with 0.2% Triton X-100
and 1% normal goat serum (NGS) in PBS overnight at 4°C. Tissue was
washed in PBS and then incubated with VECTISTAIN elite ABC reagent
(Vector Laboratories) overnight at 4°C. The tissue was washed in PBS
and then reacted with diaminobenzidine tetrahydrochloride substrate
(DAB; Vector Laboratories) for 10-15 min at RT. Sections were rinsed
in PBS and cleared with 100% glycerol for viewing with a Nikon Eclipse
E-400 microscope. Figure 1, B and C, provides
examples of intracellularly labeled cells, as well as camera lucida
reconstructions of cells located within neocortical ectopias of adult
NXSM-D/Ei and NZB/BlNJ mice. In some cases 1:2,000 dilution of
4,6-diamino-2-phenylindole (DAPI) in PBS was applied for 5 min to
visualize the ectopia as well as the disruption the ectopia causes
to the underlying cortical layers (Fig. 1A).
Immunocytochemistry
Mice were transcardially perfused with PBS followed by 4% paraformaldehyde. Brains were postfixed overnight at 4°C, blocked in 1.5-1.9% agar, and sectioned in the coronal plane on a vibratome. Free-floating sections (50 µm) were then collected into different wells for immunocytochemistry. Fixed sections were first washed with PBS and then preblocked in 5% NGS and 0.2% Triton X-100 for 1 h. Sections were incubated with rabbit anti-GABA (1:10,000) in 0.1% Triton X-100, 2.5% NGS, and PBS at 4°C for 40 h. The tissue was then rinsed several times with 2.5% NGS in PBS and then incubated in biotinylated goat-anti-rabbit (1:200) secondary antibody for 2 h at RT. Sections were rinsed several times with PBS and incubated for 1 h in an avidin and biotinylated horseradish peroxidase mixture. The tissue was rinsed in PBS and then reacted with 0.05% diaminobenzidine in the presence of 0.0015% H2O2, and 0.04% nickel chloride. Sections were collected onto gelatin-coated slides, dried for several hours, and coverslipped with Cytoseal.
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RESULTS |
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Ectopias contain diverse cell types
Layer I ectopias visible in slices examined under a dissecting
microscope (Fig. 1, A and B) were present in 43%
(30/70) of all male and 41% (11/27) of all female NXSM-D/Ei (10/17
female; 27/50 male) and NZB/BlNJ mice (1/10 female; 3/20 male). On
average there was only one ectopia present per animal, with a
preference of occurrence within the somatosensory cortex. In one brain
we identified two ectopias located adjacent to each other within the
somatosensory cortex. Whole cell recordings were made from a total of
164 cells within and surrounding these ectopias. Cells within ectopias
had resting membrane potentials of 63.5 ± 0.7 mV, input
resistances of 53.1 ± 10.0 M
, and fired multiple action potentials to depolarizing stimuli.
Cells in normatopic neocortex exhibit diverse action potential patterns
associated with different neuronal cell types. These types include
regular spiking cells, typically associated with pyramidal cells,
bursting cells, characteristic of some layer 5 cells, and a variety of
fast-spiking interneuron types (Agmon and Connors 1989;
Cauli et al. 1997
; Gupta et al. 2000
;
Kawaguchi 1993
; Kawaguchi and Kubota
1993
; McCormick et al. 1985
). Similarly cells in
ectopias exhibited a variety of different firing patterns and
accommodation rates. The firing patterns included regular spiking
(85%; Fig. 2A),
burst-discharge accommodating (10%; Fig. 2, B and
C), and burst-discharge non-accommodating neurons (5%; Fig.
2D). A plot of the spike frequency versus depolarizing
current injection for six representative cells in ectopias, recorded at RT, further demonstrates the variety of firing properties exhibited by
neurons within ectopias (Fig. 2E). Layer I ectopias,
therefore, contain neurons with diverse cellular physiologies.
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Consistent with the electrophysiologies, the morphologies of ectopic
cells, determined by intracellular labeling with biocytin, were
diverse. Figure 1C shows several examples of camera lucida reconstructions of the cells located inside ectopias. Neurons within
ectopias can be identified as either atypically oriented spiny
pyramidal cells (Figs. 1C and 8A) or aspiny and
sparsely spiny nonpyramidal cells. The pyramidal to nonpyramidal cell
ratio was ~5:1, and the presence of significant numbers of
nonpyramidal cells was further confirmed by immunocytochemical labeling
for -aminobutyric acid (GABA; Fig. 3).
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Characterization of synaptic properties within ectopias
Extracellular stimulation of white matter beneath ectopias
reliably produced excitatory PSPs (23/23, Fig.
4A) within
ectopic neurons. Paired-pulse experiments showed that with short
inter-stimulus intervals (ISIs; 50 ms) paired-pulse facilitation was
evident (PSP2: PSP1 ratio of 1.34 ± 0.06), while ISIs of 200 ms,
or longer, produced a depression in the second PSP amplitude (Fig.
4B; n = 7). To examine the potential for
plasticity at ectopia synapses, seven cycles of 50-Hz stimuli (80 ms)
were applied at 0.1 Hz while the cells were depolarized to about 20
mV. Potentiation was defined as a 20% increase, or greater, in PSP
amplitude immediately following tetanization, which was sustained for
10 min. Potentiation was elicited in one of four cells tested when
the stimulating electrode was placed in layer IV of the neocortex (Fig.
4C), and zero of nine cells tested when the stimulating
electrode was placed in the white matter (WM)/layer VI border (Fig.
4D).
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Glutamatergic and GABAergic synapses within ectopias
To determine the types of synapses formed on cells within
ectopias, we characterized the spontaneous synaptic currents recorded from cells within ectopias. At holding potentials of 40 mV both inward and outward spontaneous synaptic currents were apparent, suggesting the presence of both excitatory and inhibitory synapses (16 of 16 cells) (Fig. 5A).
GABAA receptor blockers BMI (10 µM) and PTX (10 µM) eliminated the population of synaptic currents that were outward
at
40 mV (Fig. 5B). The remaining spontaneous synaptic
currents reversed at approximately 0 mV and could be completely blocked
by a combination of d-AP5 (50 µM) and DNQX (10 µM). Blockade of
N-methyl-D-aspartate (NMDA) receptors alone with
d-AP5 revealed AMPA/kainate (A/K) receptor-mediated synaptic events
that were linearly related to membrane potential. Representative traces
in Fig. 5 had peak amplitudes of 8.93 ± 0.69 pA, rapid decay time
constants (7.6 ± 1.7 ms at
20 mV), and conductances of 4.5 ± 0.4 nS (Fig. 5D). In contrast, blockade of A/K receptors revealed NMDA receptor-mediated spontaneous synaptic events that showed
voltage-dependent block at
80 mV (Fig. 5C), had peak
amplitudes of 4.82 ± 0.34 pA, had decay time constants of
15.35 ± 2.2 ms, and had conductances of 2.4 ± 0.2 nS at
20 mV. Averages of all cells tested are presented in Table
1. In addition, the A/K receptor-mediated synaptic currents occurred at higher frequencies than NMDA
receptor-mediated synaptic currents (at
20 mV: 0.3 events/s for A/K
receptor-mediated currents, and 0.17 events/s for NMDA
receptor-mediated currents).
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To characterize the GABAergic synaptic currents, a cocktail of d-AP5
(50 µM) and DNQX (10 µM) was used to block glutamatergic events.
The remaining events could be blocked by addition of PTX and BMI and
are therefore mediated by GABAA receptors. Figure 6A shows the PSCs mediated by
activation of GABAA receptors. These currents had
reversal potentials of near the calculated Cl
equilibrium potential of
65 mV (Fig. 6B), had peak
amplitudes of 13.74 ± 1.162 pA, had decay time constants of
26.8 ± 3.1 ms at 0 mV (Fig. 6C), occurred at a mean
frequency at 0 mV of 5.1 events/s, and had conductances of 0.23 ± 0.02 nS at 0 mV (Fig. 6D). Cells in ectopias, therefore,
contain both GABAergic and glutamatergic synapses.
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Ectopias receive direct excitatory inputs from normatopic cortex
To determine whether ectopias receive input from
adjacent normatopic cortex, extracellular stimulation (0.1- to 0.2-ms,
50- to 400-µA current) was delivered to multiple adjacent sites in normatopic cortex. A bipolar stimulating electrode was placed in layer
2/3, 400 µm medial or lateral to the ectopia or
300 µm
within the axon fascicles directly below the ectopia. The axon fascicles are the same as the "bundle of fibers" reported by
Sherman et al. (1990)
. Excitatory postsynaptic
potentials were consistently elicited on stimulation of all sites
surrounding the ectopias (Fig.
7A; n = 23).
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To further characterize the excitatory inputs,
GABAA blockers (BMI and PTX) were applied to the
bath solution (Fig. 7B). Evoked exhibitory postsynaptic
currents (EPSCs) had a fast initial component and a late component that
showed voltage-dependent block at 80 mV. The late component was
isolated by addition of DNQX, and it had an average peak amplitude of
9.6 ± 0.6 at
20 mV, decay time constant of 20.5 ± 2.6 ms
(Fig. 7D), and exhibited voltage-dependent block at
80 mV
(Fig. 7C). The A/K receptor-mediated component was isolated
by blocking NMDA receptors (d-AP5, 50 µM), and it reversed at 0 mV
(Fig. 7E), had an average peak amplitude of 11.5 ± 0.5 pA at
20 mV, and had rapid decay time constants of 8.0 ± 1.4 ms
at
20 mV (Fig. 7F). Antidromic responses were rarely observed (1/29; 3%) due to stimulation of the surrounding cortex. In
addition, labeled pyramidal cells outside of ectopias
were found to send axon collaterals into ectopias (Fig.
8D). Ectopias, therefore
receive direct excitatory connections from surrounding normatopic
cortex.
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Ectopias receive direct inhibitory input from normatopic cortex
When all glutamatergic transmission was blocked with DNQX and
d-AP5, monosynaptic GABAergic synaptic currents were reliably produced
by extracellular stimulation (0.1- to 0.2-ms, 50- to 400-µA current)
delivered to multiple adjacent sites in normatopic cortex (Fig.
9A). Bipolar stimulating
electrode was placed in layer 2/3, 400 µm either medial or lateral
to the ectopia or within the axon fascicles directly below the ectopia
(
300 µm). Similar to spontaneous GABAergic synaptic currents, the
evoked inhibitory postsynaptic currents (eIPSCs) had reversal
potentials of about
60 mV (Fig. 9C), had decay time
constants of 22.3 ± 1.65 ms at 0 mV (Fig. 9B), and
were sensitive to application of BMI and PTX (data not shown).
Therefore ectopias receive both direct inhibitory and excitatory
connections from surrounding normatopic cortex.
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Epileptiform discharges in ectopias
In disinhibited slices (10 µM BMI and 10 µM PTX), low depolarizing stimulation (50 µA) of WM/layer VI border elicited late epileptiform discharges (latency, 104 ± 8.7 ms) in cells within ectopias (Fig. 10A). These large discharges within ectopias were of variable latency, 46.3-174.5 ms and were associated with a large post-discharge depolarization and multiple action potentials. As expected, the latency of epileptiform discharges were much faster on heating the slice to 34-35°C (latency 20.84 ± 1.98 ms; Fig. 10B). The epileptiform discharges could be blocked with d-AP5 (Fig. 10A).
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In 23 cells recorded in slices with single ectopias at 35°C, we never observed epileptiform discharges without the addition of GABAA receptor blockers. Epileptiform discharges, however, could be elicited in the absence of GABAA blockers, in the only slice which contained two ectopias positioned adjacent to one another (Fig. 10C, inset). Extracellular stimulation (110-120 µA) of the WM/layer VI border initiated epileptiform discharges in two neurons recorded from both ectopias (Fig. 10, C and D). The discharges were of variable latency (9.9-58.9 ms; 25.57 ± 4.16), and could be blocked with d-AP5 (Fig. 10D). Therefore multiple ectopias appear to be necessary to create an epileptogenic cortical slice.
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DISCUSSION |
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Our results demonstrate that ectopias contain neurons that have a variety of intrinsic firing patterns and morphologies, have both glutamatergic and GABAergic synapses, receive direct excitatory and inhibitory input from outside of the ectopia, and can show epileptiform bursts if slices are either disinhibited or contain two ectopias. These findings are discussed with respect to properties in normatopic neocortex and to properties of other forms of neocortical dysplasias.
Ectopias contain diverse cell types
Neurons within normatopic neocortex exhibit diverse forms of
intrinsic firing patterns that are associated with different neuronal
types. Spiny pyramidal cells have been classified based on their
different patterns of regular and bursting action potential patterns
(Agmon and Connors 1989; McCormick et al.
1985
), and similarly nonpyramidal cells have been classified
into a variety of types including irregular spiking, fast spiking
(Cauli et al. 1997
; Kawaguchi 1993
;
Kawaguchi and Kubota 1993
), accommodating, non-accommodating, or stuttering (Gupta et al. 2000
).
While we did not observe cells in ectopias in all categories reported
in the literature for neocortex, we did record from cells in ectopias that had properties of regular spiking pyramidal cells, burst-discharge accommodating, and burst-discharge non-accommodating nonpyramidal cells. Interestingly, we did not record from any cells that were classic "fast spiking cells." Nevertheless the morphologies and presence of GABA-positive cells in ectopias indicate that there are
nonpyramidal interneurons within ectopias. Consistent with this,
biocytin-filled cells within ectopias could be classified as either
atypically oriented spiny pyramidal cells or sparsely spiny and aspiny
nonpyramidal cells. The ratio of pyramidal to nonpyramidal cells using
these morphological criterion was 5:1, similar to the pyramidal to
nonpyramidal cell ratio in normatopic cortex (Hendry et al.
1987
; Parnavelas et al. 1977
). The fact that no
nonpyramidal cells in ectopias showed a classic fast-spiking non-accommodating pattern of activity could indicate that only a subset
of interneuron types reside within ectopias or that the development of
the physiology of interneurons within ectopias is altered.
Excitatory synapses within ectopias
The two main types of synapses made by neurons within normatopic
cortex, excitatory glutamatergic synapses and GABAergic synapses, are
both present on neurons within ectopias. Moreover similar to synapses
described in normatopic cortex and hippocampus in rats and mice, cells
within ectopias have glutamatergic synapses that are mediated by both
A/K and NMDA receptor activation (Fleidervish et al.
1998; Hablitz and Sutor 1990
; Hestrin
1993
; Hestrin et al. 1990a
,b
; Konnerth et
al. 1990
; LoTurco et al. 1990
; Sutor and
Hablitz 1989
). The A/K receptor-mediated synapses within
ectopias have a linear current-voltage relationship, DNQX sensitivity, and rapid decay time constants similar to normatopic cortical and
hippocampal neurons (Fleidervish et al. 1998
;
Hestrin 1993
; Hestrin et al. 1990
;
LoTurco et al. 1990
). NMDA receptor-mediated synapses
within ectopias have nonlinear current-voltage relationships, d-AP5
sensitivity, and slow decay time constants similar to cortical and
hippocampal neurons (Berretta and Jones 1996
;
Hablitz and Sutor 1990
; Konnerth et al.
1990
; LoTurco et al. 1990
; Sutor and Hablitz 1989
). Similarly recordings of supragranular cells in normatopic cortex in NXSM-D/Ei and NZB/BlNJ mice, which provide a more
direct comparison of excitatory events, show that the amplitudes, decay
time constants, and frequencies of A/K and NMDA receptor-mediated events recorded within ectopias were not significantly different from
those recorded in normatopic layer II/III cells in NXSM-D/Ei and
NZB/BlNJ mice (Table 1).
Inhibitory synapses within ectopias
GABAA receptor-mediated synaptic events
within ectopias have similar properties to normatopic cortical cells.
The present results indicate that GABAA
receptor-mediated synaptic events within ectopias have similar peak
amplitudes (range of 5-156 pA) to those reported for cortical cells of
supragranular and infragranular layers (Ling and Bernardo
1999; Salin and Prince 1996
). The decay kinetics
of GABAA receptor-mediated synaptic events within
ectopias (27 ms) are similar to those reported for cortical and
hippocampal cells at similar recording temperatures (Galarreta
and Hestrin 1997
; Ropert et al. 1990
;
Zhou and Hablitz 1997
), but as expected were
significantly slower than spontaneous IPSCs (sIPSCs) recorded at higher temperatures (Ling and Bernardo 1999
;
Otis and Mody 1992
; Otis et al. 1991
;
Salin and Prince 1996
). Recordings of supragranular
cells in normatopic cortex in NXSM-D/Ei and NZB/BlNJ mice show that the
amplitudes, decay time constants, and frequencies of
GABAA-receptor-mediated events recorded within
ectopias were not significantly different from those recorded in
normatopic layer II/III cells in NXSM-D/Ei and NZB/BlNJ mice (Table 1).
Ectopias receive input from normatopic cortex
Extracellular stimulation of the normatopic cortex surrounding
neocortical ectopias elicited both excitatory and inhibitory postsynaptic currents. Evoked EPSCs had both a fast, initial A/K receptor-mediated component and a late, NMDA receptor-mediated component that showed voltage-dependent block at 80 mV. Antidromic responses were rarely observed due to stimulation of the surrounding cortex, and pyramidal cells outside of ectopias were observed to send
axon collaterals into ectopias; therefore ectopias receive direct
excitatory connections from surrounding normatopic cortex. In the
absence of excitatory transmission, IPSCs were elicited by
extracellular stimulation of the normatopic cortex surrounding the
ectopias. The evoked GABAA receptor-mediated
events were sensitive to GABAA receptor blockers,
BMI and PTX, and exhibited a current-voltage relationship similar to
sIPSCs recorded within ectopias. Therefore ectopias also receive direct
inhibitory connections from surrounding normatopic cortex.
Although our data suggest that cells within layer I neocortical
ectopias receive input, other intracortical connections may be
disrupted by the presence of ectopias. Histochemical analysis of
ectopia cells injected with biocytin suggests that axon collaterals of
cells within ectopias are restricted by the boundaries of the ectopia
(Fig. 8C), although neurons within ectopias can extend axons
along the axon fascicles extending from the base of the malformation
(Fig. 8, B and C). These data suggest that
neurons within ectopias may be making aberrant cortico-cortical or
subcortical connections. Consistent with this hypothesis, studies using
anterograde and retrograde tracers have revealed aberrant
thalamo-cortical and cortico-cortical connections (Jenner et al.
2000); however, the functionality of these connections has not
yet been elucidated. Based on the considerable amount of evidence
suggesting a disruption of normal circuitry, further
electrophysiological analysis is needed to determine whether ectopias
disrupt normal cortico-cortical and subcortical circuitry.
Comparison to other cortical dysplasias
Heterotopias, such as subcortical band heterotopias (SBH), large
collection of ectopic neurons located below the normatopic neocortex in
humans, and microgyria, consisting of a four-layered cortex, have been
associated with the occurrence of epilepsy in humans (Chugani et
al. 1993; Fusco et al. 1992
; Lee et al.
1997
; Meencke and Janz 1984
; Palmini et
al. 1991a
,b
). Animal models of both SBH and microgyria have
provided evidence that normatopic neurons, rather than heterotopic
neurons, are responsible for initiating epileptiform activity
(Chen et al. 2000
; Jacobs et al. 1996
,
1999
; Luhmann and Raabe 1996
). In contrast,
slices with single ectopias were never found to be epileptogenic either
within or outside of the ectopia.
It has been suggested that the hyperexcitability of cells surrounding
microgyria in rats is due in part to an imbalance of excitation and
inhibition (Jacobs et al. 1996; Luhmann et al. 1998
; Prince and Jacobs 1998
; Redecker et
al. 2000
). This imbalance has been characterized by an increase
in sIPSC frequency and amplitude (Prince and Jacobs
1998
) and a down regulation of GABAA
receptor subunits (Redecker et al. 2000
), as well as an
increase in NMDA receptor-mediated input on pyramidal cells
(Luhmann et al. 1998
) and an increase of NR2B-containing
receptors (Defazio and Hablitz 2000
) within the
epileptogenic cortex of animals with induced microgyria. In contrast,
in slices with single ectopias there is no significant difference in
the frequency of excitatory or inhibitory events within ectopias as
compared with the surrounding normatopic cortex. Epileptiform-like
discharges were recorded in two neurons within ectopias in the only
slice that contained two adjacent ectopias. This suggests that the
presence of multiple ectopias may disrupt the normal balance of
inhibition and excitation, thereby creating a hyperexcitable
environment. Microgyria and SBHs generally result in much larger
cortical disruptions than a single ectopia, indicating that there may
be a critical amount of dysplasia necessary to create epileptiform activity.
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ACKNOWLEDGMENTS |
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This work was supported by National Institute of Child Health and Human Development Grant HD-20806 to J. J. LoTurco.
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FOOTNOTES |
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Address for reprint requests: J. J. LoTurco, 3107 Horsebarn Hill Rd., U-156, Dept. of Physiology and Neurobiology, University of Connecticut, Storrs, CT 06269 (E-mail: loturco{at}oracle.pnb.uconn.edu).
Received 23 August 2000; accepted in final form 24 October 2000.
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REFERENCES |
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