Low-Voltage Activated T-Type Calcium Currents Are Differently Expressed in Superficial and Deep Layers of Guinea PigPiriform Cortex
Jacopo Magistretti and
Marco de Curtis
Department of Experimental Neurophysiology, Istituto Nazionale Neurologico Carlo Besta, 20133 Milano, Italy
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ABSTRACT |
Magistretti, Jacopo and Marco de Curtis. Low-voltage activated T-type calcium currents are differently expressed in superficial and deep layers of guinea pig piriform cortex. J. Neurophysiol. 79: 808-816, 1998. A variety of voltage-dependent calcium conductances are known to control neuronal excitability by boosting peripheral synaptic potentials and by shaping neuronal firing patterns. The existence and functional significance of a differential expression of low- and high-voltage activated (LVA and HVA, respectively) calcium currents in subpopulations of neurons, acutely isolated from different layers of the guinea pig piriform cortex, were investigated with the whole cell variant of the patch-clamp technique. Calcium currents were recorded from pyramidal and multipolar neurons dissociated from layers II, III, and IV. Average membrane capacitance was larger in layer IV cells [13.1 ± 6.2 (SD) pF] than in neurons from layers II and III (8.6 ± 2.8 and 7.9 ± 3.1 pF, respectively). Neurons from all layers showed HVA calcium currents with an activation voltage range positive to
40 mV. Neurons dissociated from layers III and IV showed an LVA calcium current with the biophysical properties of a T-type conductance. Such a current displayed the following characteristics: 1) showed maximal amplitude of 11-16 pA/pF at
30 mV, 2) inactivated rapidly with a time constant of ~22 ms at
30 mV, and 3) was completely steady-state inactivated at
60 mV. Only a subpopulation of layer II neurons (group 2 cells; circa 18%) displayed an LVA calcium current similar to that observed in deep layers. The general properties of layer II-group 2 cells were otherwise identical to those of group 1 neurons. The present study demonstrates that LVA calcium currents are differentially expressed in neurons acutely dissociated from distinct layers of the guinea pig piriform cortex.
 |
INTRODUCTION |
Ion channels operated by cell-membrane voltage are known to control electroresponsiveness of neurons by regulating the efficacy of postsynaptic potentials and by setting the neuronal output modalities. In particular, voltage-dependent calcium conductances influence excitability properties of central neurons by shaping their firing and by promoting intracellular propagation of excitation between dendrites and soma and vice versa (Llinás 1988
). Moreover, calcium influx through voltage-gated channels controls several biochemical processes and regulates the expression of genes involved in survival, development, and plasticity changes of neurons (Ghosh and Greenberg 1995
). Two main classes of calcium currents with different biophysical and pharmacological properties have been described in neurons of the mammalian nervous system (Tsien et al. 1988
), i.e. high-voltage activated (HVA) and low-voltage activated (LVA) currents. Both types of currents and the underlying ion channels have been demonstrated in pyramidal neurons of the hippocampus (Fisher et al. 1990
; Kay and Wong 1987
; Magee and Johnston 1995
; Mogul and Fox 1991
) and in neurons from deep layers of the neocortex (de la Peña and Geijo-Barriento 1996; Friedmann and Gutnick 1987; Sayer et al. 1990
; Sutor and Zieglgansberger 1987
). The specific functional roles of such conductances depend on a variety of factors including their specific biophysical properties, their location in the different neuronal membrane compartments, and their possible spatial association with other conductances. It is currently believed that HVA currents are mostly involved in synaptic release (McCleskey 1994
; Takahashi and Momiyama 1993
) and in the control of other cell-function regulatory mechanisms by promoting calcium fluxes and prominent intracellular calcium-concentration increases, whereas LVA currents are implicated in dendritic amplification of distal synaptic inputs (Magee and Johnston, 1995
; Huguenard 1996
) as well as in the generation of regenerative spikes underlying somatic burst firing (Huguenard 1996
; Jahnsen and Llinás 1984
; Llinás and Yarom 1981
).
Although LVA calcium currents have been studied in several cell systems over many years, their properties and expression in mammalian neocortex and paleocortex neurons are incompletely characterized. This may be due to the following: 1) the small amplitude of LVA currents in comparison to HVA calcium currents; 2) their relatively rapid kinetics, which cause space-clamp problems in branched cells such as cortical neurons; and 3) the lack of satisfactorily specific pharmacological blockers.
The differential expression of HVA and LVA calcium conductances in distinct populations of neurons within the same cortical region, is believed to underlie specific intrinsic firing patterns and characteristic synaptic integrative properties that influence the dynamic network organization of the cortex, thereby affecting its information-processing function (Connors and Gutnick 1990
). The present study investigates the possibility of a nonhomogeneous expression of calcium currents in different subpopulations of cortical neurons. The piriform cortex was chosen as model for this study because it is composed of three unusually distinct cellular layers that can be selectively isolated for preparing acutely dissociated neurons, and because the presence of a low-threshold, calcium-dependent potential in deep but not in superficial neurons was reported in an in vitro study performed on slices (Tseng and Haberly 1989
). The analysis of calcium currents with the whole cell variant of the patch-clamp technique in neurons acutely dissociated from layers II, III, and IV of the guinea pig anterior piriform cortex demonstrated that LVA calcium currents are selectively expressed in deep layers and in a small subpopulation of layer II neurons. Part of the results have been communicated in abstract form (Magistretti and de Curtis 1996
).
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METHODS |
Cell preparation
Female Hartley guinea pigs (7-38 days old) were anesthetized with an intraperitoneal injection of ketamine (200-250 mg/kg) and decapitated. The brain was quickly extracted under hypothermic conditions and submerged in an ice-cold solution (dissection buffer) composed of (in mmol/l): 115 NaCl, 3 KCl, 3 MgCl2, 0.2 CaCl2, 20 piperazine-N,N
-bis(2-ethanesulphonic acid) -1.5 Na (PIPES-Na), and 25 glucose (pH 7.4 with NaOH, bubbled with pure O2). The two hemispheres were separated and cut with a McIlwain tissue chopper into 500-µm-thick slices. The section plane was normal to the longitudinal axis of the lateral olfactory tract. Slices that included the anterior piriform cortex were transferred into a 90-mm-diameter Petri dish coated with Sylastic (Dow Corning) and filled with ice-cold dissection buffer. The examination of the sliced fresh tissue at low magnification (×25-40) allowed easy recognition of the lamination of the piriform cortex (Fig.1A). In each experiment one of the layers (either II, III, or IV) was dissected from each slice under microscopic control. The tissue fragments obtained were then transferred into a 20-ml stirring flask filled with the dissection buffer added with 1 mg/ml pronase (protease type XIV, Sigma, St. Louis, MO) and continuously bubbled with O2. The flask was submerged in a thermostated bath at 34°C and gently stirred for 15 min. The enzymatic reaction was stopped by removing the solution and by rinsing the tissue three times with a solution (dissociation buffer) containing (in mmol/l): 113.5 NaCl, 3 KCl, 3 MgCl2, 20 PIPES-Na, 3 ethylene glycol-bis (
-aminoethyl ether)-N,N,N
,N
-tetraacetic acid (EGTA), and 25 glucose and also 2 mg/ml bovine serum albumin (Sigma fraction V) (pH 7.4 with NaOH). The tissue fragments were then placed in a holding chamber kept at room temperature and filled with a continuously oxygenated perfusion buffer, composed of (in mmol/l): 130 NaCl, 3 KCl, 2 MgCl2, 2 CaCl2, 10 N-2-hydroxyethylpiperazine-N
-2-ethanesulphonic acid (HEPES), and 25 glucose (pH 7.4 with NaOH). When needed, tissue fragments were removed from the chamber, resuspended in 2 ml dissociation buffer, and triturated with a few passages through Pasteur pipettes of progressively decreasing tip diameter. After sedimentation of the undissociated tissue, the supernatant was transferred into the recording chamber, on a concanavaline A (Sigma type V)-coated, 15-mm-diam round coverslip. The dissociated cells were allowed to settle down for 15 min before starting the recordings.

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| FIG. 1.
A: microphotograph of a fresh, 500-µm coronal section of the anterior piriform cortex as it appears during the dissection. Right: schematic drawing of the section with borders of the layers. RS, rhinal sulcus; LOT, lateral olfactory tract. Bottom: typical pyramidal cells from layer II (B and C) and multipolar neurons from layer IV (D and E) as they appear after the enzymatic dissociation.
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The recording chamber was mounted on the stage of an Axiovert 100 microscope (Zeiss, Oberkochen, Germany), and the cells were observed at ×400 magnification. Recordings were performed exclusively from neurons with a recognizable pyramidal or multipolar shape. The morphology of the cells was pyramidal-like in the case of layer II (Fig. 1, B and C) and multipolar-like in the case of layer IV (Fig. 1, C and D). Cells from layer III had either pyramidal-like or multipolar shape (not shown).
Patch-clamp recordings
The recording chamber was initially perfused with oxygenated perfusion buffer and then, after the wash-out of cell debris, with an oxygenated extracellular solution suitable for isolation of calcium currents, containing (in mmol/l) 88 choline-Cl, 40 tetraethylammonium (TEA)-Cl, 3 KCl, 2 MgCl2, 5 CaCl2, 3 CsCl, 10 HEPES, 5 4-aminopyridine, and 25 glucose (pH 7.4 with HCl). Perfusion rate was about 0.5 ml/min. Patch pipettes were fabricated from thick-wall borosilicate glass capillaries (GC 150-7.5, Clark Electromedical Instruments, Reading, UK) by means of a SutterP-87 puller (Sutter Instruments, Novato, CA). The pipette solution contained (in mmol/l) 78 Cs methanesulphonate (CsMeSO3, obtained by neutralizing CsOH with equimolar methanesulphonic acid), 40 TEA-Cl, 10 HEPES, 10 EGTA, 20 phosphocreatine di-Tris salt (PC), 2 adenosine 5
-triphosphate (ATP)-Mg, 0.2 guanosine 5
-triphosphate (GTP)-Na, and 1 adenosine 3
,5
-cyclic monophosphate (cAMP) as well as 20 U/ml creatinephosphokinase (CPK) (pH adjusted to 7.2 with TEA-OH). In some experiments on layer IV and layer III neurons, PC was substituted by equimolar CsMeSO3 and ATP, cAMP, GTP, and CPK were omitted to accelerate the rundown of HVA calcium currents, that under these conditions was complete in <10 min. No differences in LVA calcium-current voltage dependence and kinetics were noticed with the two intracellular solutions. The patch pipettes had a resistance of 4-6 M
when filled with the above solutions. Tight seals (>1 G
) and the whole cell configuration were obtained according to the standard technique (Hamill et al. 1981
). Voltage-clamp recordings were performed at room temperature (22°C) by means of an Axopatch 1-D amplifier (Axon Instruments, Foster City, CA). Cell capacitance was estimated by reading out the cell capacitance value after cancelling the whole cell capacitive transients evoked by a 10-mV hyperpolarizing voltage step with the amplifier compensation section. Series resistance (usually ~12-18 M
and always <25 M
) was routinely compensated by 50-70%. Voltage protocols were commanded and current signals were acquired with a 486 PC connected to an Axon DigiData 1200 interface, using the Clampex program of the pClamp 6.0.2 software (Axon Instruments). In all recordings the general holding potential was
70 mV. Current signals were filtered at 5 kHz, digitized at 20-50 kHz and leak subtracted via an on-line P/4 protocol.
Data analysis
Current traces were analyzed by means of the Clampfit program. Times to peak were measured from the onset of command voltage steps. Calcium permeability (PCa) was calculated from peak current amplitudes (ICa) by applying the constant-field equation in the form
in which the nominal intra- and extracellular calcium concentration values (0 and 5 mM, respectively) were introduced. Data were fitted to exponential functions using Clampfit or to Boltzmann functions (1/{1 + exp[(V
V1/2)/k]}) using Origin 3.06 (MicroCal Software, Northampton, MA). Average values were expressed as mean ± SD, and statistical significance was evaluated by means of the two-tail Student's t test for unpaired data.
 |
RESULTS |
Two morphologically distinct, but electrophysiologically homogeneous, types of principal neurons with small pyramidal and semilunar somata have been described in layer II (Haberly 1983
). We probably recorded from both types of cells, because the size and shape of their somata could not be distinguished after the dissociation procedure. Pyramidal and multipolar neurons are found in layer III, whereas in layer IV (also termed endopiriform nucleus) large multipolar neurons represent the only morphological type of principal cell (Haberly 1983
). No recordings were performed in the present study from bipolar cells or other small neurons interpretable as interneurons. Voltage-activated calcium currents were recorded in 39 neurons from anterior piriform cortex layer II, 12 neurons from layer III, and 29 neurons from layer IV. Different postnatal (P) age groups were similarly represented in the neurons from different layers: for P7-P16, P17-P26, P27-P36, and P37-P38 age groups the number of layer II and layers III-IV cells was 20 and 19, 11 and 13, 5 and 7, and 3 and 2, respectively.
Whole cell calcium currents show layer-specific expression of an LVA transient component
Typical whole cell voltage-dependent currents recorded from layers II, III, and IV neurons with our intra- and extracellular solutions are shown in Fig.2. The superfusion with 200 µM CdCl2 abolished these currents (not shown), which therefore were identified as calcium currents. In most layer II neurons (Fig. 2, top left panels) calcium currents could be evoked with test depolarizing voltage steps to
40 mV or above, whereas in all layer III and IV (Fig. 2, bottom left and right panels, respectively) and in a small percentage of layer II cells (Fig. 2, top left panel) the threshold for calcium-current activation was much lower, around
60 mV. In all cases the current-voltage (I-V) relationships (Fig. 2) showed a peak between 0 and +10 mV. Layer III and IV neurons showed a prominent shoulder in the peak-amplitude I-V (
) between
60 and
20 mV, no longer visible when the I-V relationship was constructed by measuring the current amplitude at the end of a 300-ms voltage pulse (
). The shoulder at negative potentials in the I-V curve was not observed in most layer II neurons. Only in a minority of layer II cells (top right panel in Fig. 2) the I-V relationship closely resembled that of layer III and layer IV cells. No differences in current expression were observed among different postnatal age groups (see previous section).

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| FIG. 2.
Representative whole cell calcium currents in layer II, III, and IV neurons. Top of each panel: whole cell voltage-activated calcium currents recorded in typical neurons from layers II (top right and left), III, and IV. Bottom of each panel: I-V relationships in the same cell obtained by measuring the current amplitude both at the peak ( ) and at 300 ms from the beginning of the depolarizing pulse ( ). The voltage protocol is shown in the top right panel. The test potential steps were preceded by a 500-ms prepulse at 100 mV (the holding potential was 70 mV).
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| FIG. 3.
Time courses of whole cell calcium currents in layer II and IV neurons. A: total voltage-activated calcium-current traces recorded at different test potentials (T.P.) in 2 neurons from layer II (left and middle) and one from layer IV (right). and , time points at which the peak amplitude (Ip) and the amplitude at 120 ms past the peak (I120) were respectivelymeasured at the test potential of 40 mV. B: scatter plots of the time-to-peak (ttp) as a function of the ratio I120/Ip(=R120/p)of the currents recorded at 40 mV in layer II, III, and IV neurons. In 3 additional cells from layer II no detectable calcium currents were present at the T.P. of 40 mV. , , and , experimental points obtained from the sample traces shownin A.
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Figure 3A shows typical whole cell calcium-current traces from three other representative layer II and IV neurons. In the majority of layer II cells (left traces) a detectable current appeared at
40 mV, whereas in a small number of layer II cells (middle traces) and in all layer IV neurons (right traces) a calcium current was already present at
60 mV and became fast-decaying at more positive potentials. Layer III neurons showed currents comparable to those of layer IV cells. As a first screening we analyzed the whole cell calcium currents recorded at
40 mV: for each cell we measured the current peak amplitude (Ip), the time-to-peak (ttp), and the current amplitude 120 ms past the ttp (I120). The ratio between I120 and Ip (R120/p) was calculated and taken as an inverse index of current decay. The ttp was then plotted for each cell as a function of R120/p (Fig. 3B). In all neurons from layers III (n = 12) and IV (n = 29) both ttp and R120/p fell below 20 ms and 0.3, respectively, with the ttp fluctuating around an average value of about 12 ms. On the contrary, fixing the same two limit values in the graphic relative to layer II neurons (- - -) allowed identification of two cell groups: a large majority of neurons (n = 33) in which both ttp and R120/p were higher, and a minority of neurons (n = 6) in which both ttp and R120/p were lower than the two limit values. This result means that most layer II neurons had calcium currents more slowly activating and less completely decaying than observed in layers III-IV neurons, whereas in a few layer II cells calcium current activation and inactivation rates were comparable to those of deep-layer neurons. We preliminarily distinguished between layer II neurons having ttp and R120/p higher or lower than 20 ms and 0.3, respectively, and referred to the first as group 1 neurons and to the second as group 2 neurons. An example of calcium currents from a layer II-group 2 neuron is shown in the middle column of Fig. 3A.

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| FIG. 4.
Layer-specific isolation of T-type currents via subtraction procedure. The currents evoked by voltage steps preceded by a 500-ms conditioning prepulse (C.P.) at either 100 mV (A) or 60 mV (B) in representative neurons from layer II-group 1, layer II-group 2, layer III, and layer IV are shown. A-B: traces resulting from the subtraction of the currents preceded by the 60 mV C.P. from those preceded by the 100 mV C.P. The currents obtained at 3 voltage test pulses ( 50, 30, and 10 mV, as illustrated at the bottom) are superimposed. Right column: average, normalized I-V relationships of the currents used for and yielded by the subtraction procedure in neurons from the different piriform-cortex layers. In each graphic the mean peak amplitudes of the currents obtained with a C.P. at either 100 mV ( ) or 60 mV ( ), and of the currents returned by the subtraction ( ) are illustrated. For each cell the current values have been normalized to the absolute value of the maximal observed one (I). The number of observations is 13 for layer II-group 1, 3 for layer II-group 2, 4 for layer III, and 7 for layer IV. SDs (always <20% of the mean) have been omitted for clarity.
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| FIG. 5.
T-current voltage dependence of activation and steady-state inactivation in neurons from different piriform-cortex layers. A: steady-state inactivation protocol (bottom) and current traces obtained in a representative layer IV neuron (top). The duration of the conditioning prepulses was 500 ms. B: activation protocol (bottom) and current traces obtained in another representative layer IV neuron (top). The maximal amplitude of the tail currents was measured and used to construct activation curves. C: plots of average steady-state inactivation ( ) and activation ( , ) of T-type currents in layer II-group 2 (left), layer III (middle), and layer IV (right) neurons. The activation graphics were obtained either by using the tail-current protocol shown in B ( ) or by applying the constant-field equation to the isolated T-current peak amplitude, as explained in METHODS ( ). The correspondence of the data yielded by the 2 procedures is illustrated for layer IV neurons (right). Each current (or permeability) was normalized to the maximal value of the corresponding graphic. The number of observations is 3 (inactivation) and 4 (activation) for layer II-group 2, 4 (inactivation) and 6 (activation) for layer III, and 16 (inactivation) and 10 (activation, ) or 21 (activation, ) for layer IV. Smooth lines represent Boltzmann fits to the average data. The fitting parameters for inactivation were as follows (in mV): V1/2 = 85.3, k = 7.6 (layer II-group 2);V1/2 = 86.7, k =6.9 (layer III); V1/2 = 86.5, k = 6.4 (layer IV). The fitting parameters for activation were as follows (in mV): V1/2 = 43.7, k = 6.0 (layer II-group 2); V1/2 = 43.6, k = 5.9 (layer III); V1/2 = 46.2, k = 6.7 (layer IV, ); V1/2 = 46.2, k = 6.8 (layer IV, : fit not shown).
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The existence of a prominent, transient current component in layer IV and III, but not in most layer II neurons, was confirmed by estimating the amplitude of such component as the difference Ip
I120 at the test potential
40 mV. As summarized in Table1, this quantity (normalized to the cell capacitance) was much higher in layer IV, layer III, and layer II-group 2 neurons than in layer II-group 1 neurons, whereas I120 was not significantly different in the four groups. Table 1 also shows that the average cell capacitance was significantly higher in layer IV than in layers II and III neurons, consistent with results reported by Banks et al. (1996)
for in situ piriform-cortex neurons. On the contrary, the cell capacitance was not significantly different in group 1 and group 2 neurons from layer II. These results strongly suggest that neurons from piriform-cortex deep layers express an LVA T-type calcium current, which is not observed in a large subpopulation of layer II neurons.
Layer-specific isolation of an LVA transient calcium current
To confirm the above conclusion we performed experiments in which the test pulses were preceded by a 500-ms prepulse either at
100 or at
60 mV. The traces obtained with the prepulse at
60 mV were subtracted from those recorded after the prepulse at
100 mV. The results of this procedure in four representative cells are shown in Fig. 4. The subtraction returned very little if any current in layer II-group 1 neurons, whereas an evident fast-decaying current in layer IV, layer III, and layer II-group 2 neurons was demonstrated (traces a-b in Fig. 4). The average I-V relationships of the currents used for the subtraction (
and
) and of those obtained after the subtraction (
) are illustrated in the right column of Fig. 4. In layer IV, layer III, and layer II-group 2 neurons the I-V relationship of the currents yielded by the subtraction had a threshold at about
60 mV, just like the I-V of total currents, and peaked around
30 mV. In layer II-group 1 neurons the subtraction returned tiny currents whose I-V had both threshold and peak at much more positive potentials. The peak amplitudes of the isolated LVA currents at
30 mV were 11.8 ± 6.6 (SD) pA/pF (layer II-group 2 neurons), 16.1 ± 7.0 pA/pF (layer III neurons), and 11.2 ± 5.3 pA/pF (layer IV neurons). Because it is known that LVA T-type calcium currents are steady-state inactivated at relatively negative potentials, these data confirm that layers IV and III neurons express an LVA T-type current that is missing in most layer II neurons.

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| FIG. 6.
Voltage dependence of T-current decay rate in neurons from different piriform-cortex layers. A: current traces and superimposed exponential fits from a representative layer III neuron in which the high voltage-activated calcium currents were allowed to run down; the time constants ( dec) for each T.P. are indicated. The vertical bars mark the beginning of the current region that was considered for the fitting. B: average graphics of the voltage dependence of the T-current decay time constants in layer II-group 2 ( ; n = 5), layer III ( ; n = 4-6), and layer IV ( ; n = 10-18) neurons. indicate dec values of the currents obtained after the subtraction procedure in layer II-group 1 neurons (n = 10).
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Voltage dependence and kinetic properties of LVA T-type currents in the different layers
We further characterized the kinetic properties of this current in layer IV, layer III, and layer II-group 2 neurons. The current was isolated either by the subtraction procedure or after waiting for the complete run-down of high-voltage activated currents (see METHODS). The voltage dependence of steady-state inactivation was studied by means of a standard voltage protocol (Fig.5A). The voltage dependence of activation was analyzed either by means of a tail-current protocol (Fig. 5B) or by deriving permeability values from the peak-current amplitudes (see METHODS). Figure 5 shows current traces recorded in two representative layer IV neurons (A and B) and the average graphics for layer II-group 2, layer III, and layer IV cells (left to right in C, respectively). In all of the three groups of neurons the steady-state inactivation was complete at
60 mV, whereas the activation began near
60 mV and was nearly maximal at
25 mV.
Figure 6 shows the voltage dependence of the current decay rate. The decay part of the currents could be fitted by a single exponential, with a time constant that became progressively faster at more positive potentials (Fig.6A). Panel B shows the average voltage dependence of the decay time constant (
dec) for layer II-group 2, layer III, and layer IV neuron currents (
,
, and
, respectively). In all three neuronal groups
dec displayed a marked voltage dependence, falling from ~90 ms at
60 mV to ~22 ms at
30 mV or above. All these features are typical of a T-type current. On the contrary, in layer II-group 1 neurons, when the above described subtraction procedure returned measurable currents, these currents showed much slower decay time constants (~120 ms at
30 mV: Fig. 6B,
).
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DISCUSSION |
The present study demonstrates that LVA T-type calcium currents are segregated in subpopulations of principal neurons in the guinea pig piriform cortex. The experimental preparation utilized, namely the acutely isolated neuron, allowed satisfactory voltage-clamp conditions of the fast LVA current, because most of the dendritic arborization, with the exclusion of the large proximal dendrites, was eliminated by the dissociation procedure. We found an LVA calcium conductance in all freshly dissociated pyramidal and multipolar neurons of the deep layers III and IV. In the large majority of layer II neurons (group 1) no measurable LVA current was present; only a small subpopulation (group 2) represented by 18% of layer II cells displayed an LVA current similar to that observed in deep-layer cells. The biophysical properties of the LVA calcium current in piriform-cortex cells are similar to the T-type calcium current previously described in sensory neurons (Carbone and Lux 1984), in thalamic neurons (Coulter et al. 1989
; Hernandez-Cruz and Pape 1989
), in pyramidal cells and interneurons of the cornu Ammonis in the hippocampus (Fraser and McVicar 1991; Kay and Wong 1987
; Mogul et al 1991
), and in pyramidal neurons of the neocortex (de la Penã and Geijo-Barriento 1996; Sayer et al. 1990
; Sutor and Zieglgansberger 1987
).
Because we found LVA currents in a minority of layer II neurons, the question arises as to the specific identity of such cells. The shape of the soma and the proportion between soma and proximal dendrites did not differ among group 1 and group 2 layer II cells. The probability of observing an LVA current did not depend on the amount of available cell membrane, because the membrane capacitance was similar in the two different subpopulations of layer II cells. Finally, the possibility that the neurons endowed with LVA current could originate as a contaminant from layer III seems unlikely, because layer II was easily identified during the dissection (see METHODS) because of its much higher cell density with respect to layer III, and the boundary region between layers II and III was routinely excluded when neurons were isolated from layer II. Therefore it would appear that two subpopulations of principal cells exist in layer II, the larger one displaying no LVA currents in their soma and proximal dendrites.
Because of the use of acutely isolated neurons, the present study allowed us to ascertain the expression of an LVA current in the somatic and in the proximal dendritic compartments of piriform-cortex neurons, but did not consent to evaluate the presence of such a current in the distal dendrites, where different densities of T-type calcium conductances with respect to the somatic region were recently demonstrated in cerebellar and thalamic neurons (Budde et al. 1996
; Mouginot et al. 1996
). Nevertheless, our findings are likely to be of specific physiological significance, because neuronal LVA currents were proposed to serve different functions in somatic versus dendritic compartments: dendritic LVA currents are believed to play a role in the amplification of excitatory postsynaptic potentials (EPSPs) arising at sites electronically distant from the soma, whereas somatic LVA currents in different neuronal systems are known to participate in the generation of such events as low-threshold spikes (Llina
and Yarom 1981), burst firing (Jahnsen and Llina
1984), and depolarizing afterpotentials (Zhang et al. 1993
). Moreover, data obtained with current-clamp studies performed on piriform-cortex slices indicate that, upon rapid depolarization from resting membrane potential, deep piriform-cortex neurons discharge a low-threshold slow spike sustained by the activation of a calcium conductance (Tseng and Haberly 1989
). Such regenerative potentials were never observed in layer II neurons (Constanti and Galvan 1983
; Forti et al 1997
; Libri et al. 1994
; Tseng and Haberly 1988
). These data are consistent with a selective expression of somatic/proximal dendritic LVA calcium currents in layers III and IV and with the existence of a specific physiological role for these currents. Interestingly, preliminary data suggest that also acutely dissociated layer IV neurons recorded in current-clamp conditions are able to generate low-threshold calcium spikes when depolarized by an intracellularly injected current step, whereas layer II cells respond with a tonic, repetitive firing (unpublished observations).
Studies performed on slices demonstrated that other basic electrophysiological properties of principal piriform-cortex neurons differ substantially in superficial versus deep layers. In comparison to superficial principal cells, the pyramidal and multipolar neurons in deep layers exhibit the following characteristics: 1) higher membrane capacitance because of their larger somatic membrane surface and the more widespread dendritic arborization (Banks et al. 1996
), 2) higher input membrane resistance (Libri et al. 1994
; Tseng and Haberly 1989
), 3) generation of fast action potentials of lower amplitude and longer duration (Tseng and Haberly 1989
), and 4) endowment of a rapidly inactivating outward potassium current of smaller amplitude and slower activation and inactivation kinetics (Banks et al. 1996
). Together with the ability of deep neurons to generate low-threshold calcium spikes, some of these differences have been suggested to confer a higher intrinsic excitability to deep piriform-cortex neurons as compared to superficial ones. Our observation of a selective expression of an LVA calcium currents in layers III and IV cells is in agreement with this general conclusion, because the presence of an LVA calcium conductance can contribute in a variety of ways to enhance of the effects of sub or suprathreshold membrane depolarizations.
The question then arises of the specific functional role(s) of deep-layer LVA calcium currents and of the subthreshold depolarizations that they sustain. It has been shown that in deep piriform-cortex neurons the calcium-dependent low-threshold spike induces a fast depolarization that, unlike other types of neurons (Avanzini et al. 1989
; Jahnsen and Llina
1984; Llina
and Yarom 1981), does not generate high-frequency bursting (Tseng and Haberly 1989
). Indeed, abrupt depolarization from the resting membrane potential in deep neurons induces a fast sodium spike followed by a calcium-dependent low-threshold depolarization reminiscent of the depolarizing after-potential (DAP) described in cortical neurons (Friedman and Gutnick 1987
; Sutor and Zieglgansberger 1987
; Wong and Prince 1981
; also see Tseng and Haberly 1989
, Figs. 7 and 9). In dentate gyrus cells a DAP sustained by an LVA calcium current was proposed to boost EPSPs while transiently increasing transmembrane calcium entry (Zhang et al. 1993
). The role of LVA currents in facilitating synaptic potentials was further strengthened by the demonstration that DAPs mediated by the activation of a T-type calcium current regulate the plastic cellular changes that promote long-term potentiation of the efficacy of suprathreshold synaptic potentials in the visual cortex of kittens (Komatsu and Iwakiri 1992
).
Intrinsic bursting activity and prominent DAPs have been demonstrated in deep pyramidal cells of the neocortex (layers IV-VI) that show long apical dendrites (Connors and Gutnick 1990
; Magee et al. 1995
), whereas pyramidal neurons in the more superficial layers II and III display a regular tonic firing without DAPs (Connors and Gutnick 1990
). Moreover, in a recent study de la Peña and Geijo-Barrientos (1996)
demonstrated that the T-type LVA calcium current is expressed almost exclusively in neurons located in the deep layers of the guinea pig prefrontal cortex, whereas superficial neurons in layers II and III show neither LVA conductances nor low-threshold calcium spikes. As for other cortical areas, the results reported here confirm that LVA calcium currents are selectively expressed in deep cortical cells and possibly contribute to the basic intrinsic functions that these neurons perform differently from superficial neurons.
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FOOTNOTES |
Address for reprint requests: M. de Curtis, Dept. of Experimental Neurophysiology, Istituto Nazionale Neurologico Carlo Besta, via Celoria 11, 20133 Milan, Italy.
Received 29 April 1997; accepted in final form 17 October 1997.
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