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The ontogeny of repetitive firing and its modulation by norepinephrine in rat neocortical neurons
Det,elopmental Brain Research, 73 (1993) 213-223 © 1993 Elsevier Science Publishers B.V. All rights reserved 0165-3806/93/$06.00
213
BRESD 51635
The ontogeny of repetitive firing and its modulation by norepinephrine in rat neocortical neurons N.M. Lorenzon and R.C. Foehring Department of Anatomy and Neurobiology, The Unit'ersity of Tennessee at Memphis. Memphis, TN 38103-4~)01 ( UX4 (Accepted 22 December 1992)
Key words." Cortex; Development; Afterhyperpolarization; Norepinephrine: Repetitive firing: Modulation
The postnatal ontogeny of electrical properties was studied in rat sensorimotor cortical neurons (P6 to adult) using intraccllular recording in a n in vitro slice preparation. Many action potential properties and input resistance changed during the first 4 postnatal weeks. Repetitive firing behavior also changed during the first postnatal month. Spike-frequency adaptation was much stronger in immature neurons. At 1 week postnatal, the majority of cortical neurons would only fire for less than 200 ms regardless of the intensity of long depolarizing current injections. These cells were normal in other parameters and could fire throughout a depolarizing current injection in the presence of inorganic calcium channel blockers or norepinephrine (NE), suggesting that the inability to fire was not due to injury. The frequency with which we encountered cells with this extreme adaptation decreased with age. Spike-frequency adaptation in immature neurons appears to be primarily controlled by Ca-dependent K + conductances as in mature neurons. In mature and immature neurons, three afterhyperpolarizations (AHPs) could be distinguished by their rate of decline. The fast AHP followed repolarization of a single spike and was only partially Ca- and K-dependent. The medium duration AHP was Ca-dependent and apamin-sensitive and the slow AHP was partially Ca-dependent and not blocked by apamin. NE decreased the slow Ca-dependent AHP via /3-adrenergic receptors. This effect of NE on AHPs appeared qualitatively similar throughoul postnatal development. NE had a proportionately greater effect in younger neurons, however, due to their relatively larger slow AHP. The quantitative differences of NE's action on the slow AHP (sAHP) led to a qualitative difference in NE's effect on firing behavior. The effects of NE on firing behavior may therefore be greater during times critical for cortical maturation.
INTRODUCTION
c h a n g e d u r i n g the first p o st n at al m o n t h . Projections of t h a l a m o c o r t i c a l and callosal a f f e r e n t s c o n t i n u e to de-
A t birth, n e u r o n s
in rat s e n s o r i m o t o r c o r t e x are
v el o p postnatally 32'~3. S y n a p t o g e n e s i s c o n t i n u e s until
m o r p h o l o g i c a l l y an d electrically i m m a t u r e and several
a b o u t P28 3° In i m m a t u r e n e u r o n s , electrically e v o k e d
c h a n g e s o c c u r in the electrical p r o p e r t i e s
of t h e s e
postsynaptic p o t e n t i a l s (PSPs) are l o n g e r in latency and
n e u r o n s over the first 4 p o s t n a t a l weeks. A c t i o n p o t e n -
d u r a t i o n and are m o r e subject to failure during repeti-
tials are q u i t e b r o a d at birth, but th e i r width d e c r e a s e s
tive s t i m u l a t i o n 17. T h e relative b al an ce of synaptic exci-
t h r o u g h p o s t n a t a l wee k s 1 - 4 (the rates o f b o t h rise and
tation and inhibition also c h a n g e s with age. F o r exam-
r e p o l a r i z a t i o n increase), with the most d r a m a t i c c h a n g e s o c c u r r i n g d u r i n g the first p o s t n a t a l w e e k 17'22.
ple, d u r i n g the first p o st n at al week, synaptic inhibition is not o b s e r v e d in rat n e o c o r t e x iT.
T h e m a x i m u m rate o f rise o f Ca 2+ spikes also in-
T h e s e p o s t n a t a l m o r p h o l o g i c and synaptic c h a n g e s
c r e a s e s t h r o u g h o u t this t i m e 22. T h e s e c h a n g e s in action
parallel o n t o g e n e t i c c h a n g e s in the integrative ability
p o t e n t i a l s are t h o u g h t to r e f l e c t i n c r e a s e s in density o f
of cortical n e u r o n s .
Na +, K + and Ca 2+ c h a n n e l s d u r i n g p o s t n a t a l d e v e l o p -
r e l a t i o n s h i p b e t w e e n f r e q u e n c y and injected c u r r e n t
m e n t (c.f. refs. 10,14). I n p u t resistance, specific m e m -
(f-l)
b r a n e r e s i s t a n c e an d m e m b r a n e t i m e c o n s t a n t all dec r e a s e o v e r this 4 - w e e k p e r i o d 17'22.
a v e r a g e d u r a t i o n o f the slow a f t e r h y p e r p o l a r i z a t i o n s ( A H P s ) which follow r e p e t i t i v e firing is longer, in thc i m m a t u r e n e u r o n s 22. In adult cortical n e u r o n s ~'1~'21"2~',
T h e a f f e r e n t inputs to t h e s e cortical n e u r o n s also
has b e e n
F o r e x a m p l e , the slope of the
reported
to be less s t e e p
and
the
Correspondence: N.M. Lorenzon, Department of Anatomy and Neurobiology, 855 Monroe Avenue, The University of Tennessee at Memphis, Memphis, TN 38103-4901, USA. Fax: (1) (901) 528-7193.
214 t h e A H P s e l i c i t e d by s p i k e t r a i n s r e f l e c t c o n d u c t a n c e s
W e test t h e h y p o t h e s i s t h a t a q u a l i t a t i v e c h a n g e in the
a c t i v a t e d by t h e s u s t a i n e d d e p o l a r i z a t i o n d u r i n g firing.
m o d u l a t i o n by N E o c c u r s d u r i n g d e v e l o p m e n t .
T h e s l o w e r A H P s a r e d o m i n a t e d by C a - d e p e n d e n t
K ~ MATERIALS AND METHODS
c o n d u c t a n c e s w h i c h a r e p r i m a r i l y r e s p o n s i b l e for s p i k e frequency
adaptation <~s'2~'>. M u c h
less is k n o w n , h o w -
For the in vitro slice preparation, a block of tissue was removed from the sensorimotor cortical region of pentobarbital sodium (35 mg/kg) or metofane anesthetized rats (n = 61). The tissue sample was immediately sectioned at 400-500 p.m (for the first 2 postnatal weeks thicker sections were used; we have found this increases cell viability) in artificial cerebrospinal fluid (aCSF), containing (in mM): 125 NaCI. 3 KCI, 2 CaCI 2, 5 MgCI2, 1.2 NaH2PO4, 26 NaHCO 3, 20 dextrose, (pH = 7.4; 310-320 mOsm/I). Slices were cut at 4°C using a WPI oscillating tissue slicer. Slices were then incubated in holding chambers containing aCSF bubbled with carbogen (95% O~/5% CO 2) at 30°C for at least 1 h. Individual slices were then transferred to an interface recording chamber maintained at 34°C (tissue from immature rats was held at 34°C to improve viability ~7. Slices from adult rats were also kept at this temperature to control tk~r the temperature-dependence of AHPs>). The slice was bathed in oxygenated aCSF which flowed under the slice. Carbogen-saturated water vapor flowed over the top of the slice. To observe the effects of zero Ca :+ on cells, the Ca z+ in the aCSF was replaced with 2 mM Co 2+ and the NaH2PO 4 was omitted to avoid precipitation. Apamin (10-200 nM), NE and adrenergic agonists and antagonists were added directly to the aCSF: arterenol-bitartrate salt (25-100 /zM), isoproterenol-HCl (50-100 /xM), clonidine-HCI (100 /xM), phenylephrine-HCI (100 /xM), timolol-maleate salt (25 /xM). All drugs were purchased from Sigma except isoproterenot and clonidine (RBI). Microelectrodes (40-180 M~fl) were filled with 2 M potassium acetate, 2 M KMeSO 4, or 1% biocytin/2 M potassium acetate solutions. Tissue sections containing cells filled with bioeytin were processed histochemically using the ABC technique (modified from ref. 13; see ref. 7). All cells that were successfully filled and recovered were pyramidal cells (n = 19). No fast-spiking or intrinsic burstfiring neurons zt were included in this study. Recordings were from layers II-V (with most in layer II/1II or V). All recordings utilized an Axoclamp-llb electrometer (Axon Instruments) in continuous bridge or discontinuous current clamp
e v e r , a b o u t t h e s e p r o c e s s e s in i m m a t u r e c o r t i c a l n e u rons. In s e v e r a l a d u l t s p e c i e s , n o r e p i n e p h r i n e
(NE)
af-
fects t h e A H P s a n d firing b e h a v i o r o f c o r t i c a l n e u r o n s . F o r e x a m p l e , in a d u l t cat B e t z cells, N E r e d u c e d t h e slow A H P , i n c r e a s e d firing r a t e a n d d e c r e a s e d spikef r e q u e n c y a d a p t a t i o n s. T h e s e a c t i o n s o f N E w e r e p r o d u c e d by r e d u c t i o n o f a slow C a - d e p e n d e n t
K + con-
d u c t a n c e via /3~ r e c e p t o r s s. N E has b e e n i m p l i c a t e d as influencing ocular dominance
plasticity in d e v e l o p i n g
v i s u a l c o r t e x (e.g. refs. 2,9,15). S i n g e r a n d c o l l e a g u e s 27 h a v e p r o p o s e d t h a t for t h e s e c h a n g e s to o c c u r , a m o d i fication threshold must be reached. If NE blocked a Ca-dependent
K + conductance
in i m m a t u r e
cortical
n e u r o n s as in a d u l t n e u r o n s , t h e i n h i b i t o r y e f f e c t s o f this K + c u r r e n t w o u l d b e r e d u c e d a n d t h e p r o b a b i l i t y that
the
neuron
would
reach
such
a
modification
t h r e s h o l d w o u l d b e i n c r e a s e d . It is t h e r e f o r e i m p o r t a n t to u n d e r s t a n d
the mechanisms of action of NE during
p o s t n a t a l d e v e l o p m e n t , w h e n p l a s t i c c h a n g e s in c o r t i cal w i r i n g a r e m o s t e v i d e n t . In t h e p r e s e n t study, w e d e s c r i b e t h e o n t o g e n y o f r e p e t i t i v e firing b e h a v i o r in n e o c o r t i c a l p y r a m i d a l n e u rons and initiate studies concerning the development of modulation
of AHPs
a n d r e p e t i t i v e firing by N E .
P8
A1
P15
P29
P22
B1
C1
,.mv
D1 -72 mV -~
I
80 mV 4nA l ~ 8 ms
A2 -62 mV
-68
-78 mV
240 ms
Fig. 1. Changes in electrical properties during the first 4 postnatal weeks. A1-DI: spike width decreased with age. Action potentials elicited with 5 ms stimulus pulses. A2-D2: rheobase and input resistance traces for the first 4 postnatal weeks. Input resistance decreased, but rheobase did not change throughout postnatal development.
215 TABLE I
Electrical properties during postnatal det'elopment Values expressed as m e a n + S . D . Spike width measured at half amplitude. Rheobase is the minimum amount of current to elicit an action potential with 200 ms current injection. * indicates properties which showed significant differences between age groups in an analysis of variance test (~ = (l.05). Other symbols note significant differences with pairwise comparison using post hoc test: ~, different from P6-10: ", different from PI2 17: #, different from P20-22; o different from P27-33: +, different from adult group. Number of animals for each age group: 10 (for 6-111 days), 18 (for 12-17 days), 6 (for 20-22 days), 5 (for 27 33 days), 22 (for adult).
Electrical
Age of rat
properties
6 - 10 days
12 - 17 days
20 - 22 days
2 7 - 33 days
aduh
Resting potential (mY)
66 + 7 (24) 76.3±8.1 (26) #©+ 1.93 ± 0.75 (24) ~ # o + 182+98 (17) #c> + 49+_ 19 (17) o 0.31 +0.33 (24) 87.11 ± 46.3 (18) ~#©+
- 67 ± 6 (81) 83.0+ 13.4 (71) #:: " 1.35 _+{).56 (72) ~#o+ 219+ 109 (59) < ~ 49+ 15 (59) # c 0.32+0.33 (70) 61.8 + 30.8 (60) ~
- 68 + 6 (32) 90.3+_ 14.3 (29) ~ 0.97 + I).28 (29) ~ 274±91 (25) + 71 + 16 25) 0.30±0.14 (25) 45.8 ± 18.1 (23) ~
68 + 6 (19) 97.3+ 10.4 (21) '~ ~ 0.70 + 0.14 (21) ~'" 351 + 117 (18) ~ = # 103+36 (18) ~'' # I).27+0.16 ( 171 50.3 + 37.2 (19) ~
-- 70 + 7 (47) ~9.8± 11.3 192} " " 0.77 + (}.22 (89) ~'~' 347+ 87 (80) ~ " '~ 73+40 (8111 ~ " (}.40 + 1t. 13 (83) 42.0 + 22.8 (74) ~:~
AP amplitude (mV) * Spike width (ms) *
d V / d t , up ( V / s ) * d V / d t , down ( V / s ) * Rheobase (nA) Input resistance (M 11) *
mode. Data were recorded and stored on videotape using a Neurocorder (Neurodata) and a VCR. A H P s and firing frequency were visualized and measured off-line with RC Electronics Computerscope. The resting membrane potentials were estimated from continuous chart recordings of DC potential. Continuous monitoring of m embr ane potentials on the chart recorder allowed estimates to be made regarding drift in cell membrane potential due to electrode polarization. All statistical data are presented as mean ± S.D. Statistical differences between various age-groups for electrical properties was determined by an analysis of variance ( a = 0.05 level). A post hoc test (Tukey's HSD) was used for those measures in which a significant difference was found in the analysis of variance.
RESULTS During the first 4 postnatal weeks, several changes in neuronal electrical properties of neocortical cells were observed. Action potential width (measured at half amplitude) and input resistance decreased with age of the animal. Action potential amplitudes and the rates of rise and repolarization of action potentials increased with age (Fig. 1, Table I; see also refs. 17,22). Resting membrane potential did not vary significantly with age. In response to prolonged (500-1000 ms) current injection, adult rat cortical neurons fire repetitively throughout the stimulus (Fig. 2A). The average rate of firing increases linearly or bilinearly with increased injected current (Fig. 2B, Table II). Instantaneous firing frequency (1/interspike interval) is initially high in adult cells, but decreases with time during the stimulus (spike frequency adaptation: f-t relationship, Fig. 2D). Some cells from 1-week-old animals also fired repetitively, as in adult neurons. In these cases, the slope of
the f-1 relationship was less steep (Table It, Fig. 2B) and spike-frequency adaptation was stronger than in the adult. T h e f-I slope increased with age (f-I slope at 1 week = 25.2_+ 7.4 H z / n A (n = 51, at 2 weeks = 35.5 _+ 15.2 H z / n A (n = 101, at 3 weeks = 37.6 + 13.3 H z / n A (n = 5), at 4 weeks = 46.0 _+ 14.0 H z / n A (n = 4)). Several cells from 1-3-week-old rats had a different firing pattern; regardless of the intensity of the injected current, these cells would only fire through the first 100-250 ms of a 1-s depolarizing current injection (Fig. 2C,D). This period of firing was followed by a pronounced, prolonged slow A H P (Fig. 3D,F). In some cases, firing would resume upon termination of this A H P . Even t h o u g h these cells only fired a few spikes at
T A B L E II
Differences in firing and AHf3~' in 1-week-old and adult neocortieal neurons Repetitive firing evoked by 20-2 ms repetitive stimuli at 100 Hz at resting me mbra ne potential. # Includes only l-week-old cells tha! would fire throughout a l-s current injection. All of the above pa ra me t e rs significantly differed between 1-week-old and adult neurons (Student's t-test; a = 11.05). Resting membrane potentials did not differ significantly between age groups.
1-week-old mAHP (after 1 spike; mV) m A H P (after repetitive firing, mV) sAHP(mV) s A H P duration (s) f-lslope(Hz/nA) #
4.5 ± 3.9 10.5 + 1.2 5.6 +1.3 4.26+0.82 25.2 +7.4
Adult (22) (8) (8) (8) (5)
3.0 ±
1.6 (20)
9.1 + 1.3 1.9 ± 0.6 1.49± 0.42 57.5 ±47,7
(7) (7) (7) (10)
216 ADULT
the beginning of a depolarizing pulse, the slow AHP (sAHP) was still greater in amplitude and duration than in adult cells which fired throughout the same stimulus. The frequency with which we encountered cells with this firing pattern (100% adaptation at 300 ms during a l-s current injection) decreased with age (Fig. 2D inset). We will refer to these cells as completely adapting cells. During the first postnatal week a majority of the cells (58%) were completely adapting; 37% of the cells from 2-week-old animals and 25% of the cells from 3-week-old rats also showed this firing pattern. We did not observe completely adapting cells
-73 mV
C
/r::::
-77 rnV=-J-- -
mAH~
ADULT
P15
I[
ADULT
E
40
B
'omvI
-
~
P15
HP
I I mAHP s
F
p
P9
30-
~
loo0.2
04
0.6
-75 mV
D ;~
_~o
tJ
<
t
Z
,,~
~
2 3 4 z5 A G E (weeks)
40
o -67 m V ~
(nA)
°o 160
ms
1.0
0.8
CURRENT
.v, ~"
,
200
•
TIME
,
•
,
400
,
•
,
600
(ms)
Fig. 2. Repetitive firing behavior in adult and 1-week-old rat neocortical neurons. AI: adult regular-spiking cell fires throughout a 1-s depolarizing stimulus pulse (0.3 nA) at resting membrane potential (RP). A2: increasing the stimulus current injection (0.8 nA) increases the rate of firing. Spike amplitude clipped due to digitization. B: steady-state firing frequency plotted vs. injected current intensity. The average frequency of firing during the last 500 ms of firing is shown for the adult cell illustrated in A1, 2 (open symbols) and a P15 neuron which fired repetitively (filled symbols, different from cell in C1 and C2). The solid line through the adult data is a least squares fit with slope of 47.6 Hz/nA. CI: repetitive firing in a P15 neocortical neuron. Some immature neurons would not fire repetitively throughout a long, depolarizing current injection (0.8 nA; 100% spike-frequency adaptation by 200 ms). A spike was evoked towards the end of the stimulus pulse (probably due to sufficient decay of the prolonged AHP). C2: increasing the stimulus pulse (1.2 hA) increases the number of spikes at the beginning of the pulse, but the cell still does not fire throughout the stimulus. D: adult (open symbols, same cell as in A) and P15 (closed symbols, same cell as in C) neocortical neurons exhibit spike-frequency adaptation (0.8 nA current injection). Instantaneous firing frequency (1/interspike interval) plotted vs. time during a 1-s repetitive firing episode. The young cell stops firing within the first 100 ms (100% adaptation). Inset: the frequency with which we encountered cells with this firing pattern (100% adaptation by 300 ms) decreased with age.
Fig. 3. AHPs in adult and immature neocortical neurons. A,B: AHPs evoked by a single spike in adult (A) and a P15 cortical celt(B). A fast AHP (fAHP) could be observed as a notch or interruption in spike repolarization and a medium AHP (mAHP) follows the delayed depolarization (DD). C,D: AHPs evoked by repetitive firing episode. The mAHP immediately follows the train of spikes. The slow AHP (sAHP) follows the mAHP. Note that the sAHP in the immature neuron is much larger and longer despite the shorter duration of firing. In the immature neuron, the mAHP is evident as a brief notch before the development of the sAHP. E,F: the AHPs increased in amplitude with increasing number of spikes: AHPs from an adult neuron (E) elicited by repetitive 2 ms stimuli at 100 Elz (at resting potential (RP); 5, 10, and 30 spikes). The sAHP is relatively short and l0 spikes or more are required in order to elicit a measurable sAHP. AHPs from a P9 cell (F) were elicited by repetitive 2-ms stimuli at 100 Hz (at RP; 2, 5, 10 and 30 spikes). A sAHP is measurable after only 2 spikes and after 5 spikes appears kinetically distinct from the faster AHPs. A, C and E are all from the same cell and B and D are from another cell.
from rats older than P28. The remaining cells fired repetitively. The inability to fire repetitively throughout a current injection could indicate that the celt was injured by the impalement. Since immature neurons are typically more difficult to record from, it is important to distinguish between injured neurons and a distinct firing pattern. We only considered cells in which 100% adaptation was accompanied by a large AHP. In addition, immature cells with input resistances less than 20 MS2 or action potential amplitude less than 6 0 mV were not considered. The immature neurons in this study appeared healthy with respect to standard measures (action potential amplitude, input resistance and resting membrane potential). There were no differ-
217 pendent (data not shown; see also refs. 26,18) and increases in amplitude with multiple action potentials (Fig. 3C,E). Following a repetitive firing episode in adult rat neocortical cells, two AHPs with different rates of decay were present: a medium AHP (mAHP) and a slow AHP (sAHP). The mAHP was present after one spike (lasting 126.7 ms_+76.9 ms; n = 76) and increased in amplitude with additional spikes. Medium AHP amplitudes were measured as the peak hyperpolarization immediately following the firing episode (Fig. 3C,E, Table II). The sAHP duration decayed more slowly (over seconds), required 10-20 spikes to be elicited and was smaller in amplitude than the mAHP (Fig. 3C,E, Table II). The sAHP amplitude was measured 500 ms after cessation of the stimulus pulse (when decay of the m A H P should be nearly complete). These AHPs were similar to those previously reported in other neocortical n e u r o n s 6"18'26. To control for differences between cells from various ages in rate of firing, we elicited AHPs by evoking a set number of spikes at a constant frequency using repetitive 2-ms suprathreshold current injections. Whereas 10-20 spikes at 100 Hz were required to obtain a measurable sAHP in adult neurons, only 2-5 spikes were required in most immature neurons (Fig. 3E vs. 3F). This suggests that in younger animals the
ences in measured electrical parameters (action potential amplitude and width, input resistance, or rheobase) between those immature neurons which fired repetitively and those which were completely adapting. Stronger evidence for the health of these cells comes from the observation that these cells will fire throughout a long depolarizing stimulus pulse in the presence of inorganic Ca 2+ channel blockers (Fig. 5) or neurotransmitters (Fig. 7, see text below). The transmitter effects, in particular, are reversible (Fig. 7). Together these observations suggest that some neocortical neurons do not fire repetitively because, compared to adult cells they display a relatively greater expression of conductances underlying the sAHP. In cortical neurons of all ages, a single action potential is followed by a multiphasic afterpotential (Fig. 3A). Typically spike repolarization leads to an initial notch or pause in repolarization depending upon membrane potential. In cat 26 or human 18 neocortical neurons, this has been referred to as the fast A H P (fAHP) and is partially due to K + conductances. The fAHP is also seen as a sharp hyperpolarization during the interspike interval (ISI) during repetitive firing (Fig. 3C). After a single spike, the fAHP is followed by a delayed depolarization (DD) followed by a longer-lasting medium AHP (mAHP) or a depolarization followed by the mAHP (Fig. 3A, Table lI). This m A H P is Ca-de-
A
~
10Z
A
8
O
A
8-
I--
A
.J
~
B
A
~12
4
•
A
RA '~
0
• "o, " , 50
o .....
100
150
200
0
250
.:.!:. 7:. "., 4
8
12
AREA (mV-s)
INPUT RESISTANCE(MQ)
11 ADULT I O 1 WEEK OLD (REPETITIVELY FIRING) •1 WEEK OLD (COMPLETELY ADAPTING)
C
D D
31o z
(2_
8
•~
6¸
~ ZX ~
Q. "r
2
A A
A
:~ 0.20 ta~ ~ 0.,~
~
A
A
°/~o° o A o° AREA (mY-s)
o
0,I0
zx
A A A
z
o3
,5 0.oo
A Q
1'2
AREA (mY-s)
Fig. 4. Possible factors contributing to differences in the sAHP. A: s A H P amplitude is plotted as a function of input resistance of the cell. In all parts of this figure, open circles represent 1-week-old cells which fired repetitively, open triangles represent completely adapting 1-week-old cells and filled in squares represent adult cells. Cells with similar input resistance can have very different A H P amplitudes. B: A H P duration is correlated with the integrated area under the spikes in adult neurons (r = 0.79). C: the relationship between the A H P duration and integrated spike area differs between repetitive firing and completely adapting 1-week-old neurons. D: the relationship between A H P a m p l i t u d e / i n p u t resistance and the integrated area under the spikes in adult cells, repetitive firing immature cells and completely adapting immature cells.
218 conductances underlying the sAHP would more frequently play a role in regulating firing under physiological conditions. Since action potentials are broader and input resistance higher in immature neurons (Fig. 1, Table I), the longer A H P in immature cells could reflect a greater influx of Ca 2+ during the broader spike, with increased input resistance leading to a greater membrane voltage change in response to similar underlying A H P currents. The relative A H P amplitude was similar for adult and repetitive firing immature cells (Fig. 4A), but the A H P amplitude was consistently higher for completely adapting cells than for adult or repetitive firing immature cells at a given input resistance (Fig. 4A). Input resistance did not differ between repetitive firing and completely adapting immature neurons. To control for age-related differences in spike width, we integrated the area under the action potentials used to elicit AHPs for eight adult cells and ten neurons from 1-week-old rats (number of spikes was controlled by using a 2-ms repetitive stimuli train and 2 - 6 different spike trains with various numbers of spikes were analyzed for each cell). The area under the spikes correlated with the number of spikes in the train in both adult (r = 0.79 (20 cells)) and immature cells (r = 0.50 (31 cells)). In adult neurons, the duration of the sAHP increased with an increase in the area under the spikes (Fig. 4B). In the 1-week-old neurons, the AHP duration and area appeared to have a different
relationship than that in the adult (Fig. 4C). Repetitive firing cells from 1-week-old rats show a weak increase in AHP duration with area as in adult neurons (Fig. 4C). In contrast, completely adapting cells show all inverse relationship with the longest AHPs generated by smaller spike areas (smaller than 5 mV-s; Fig. 4C). These data suggest a different relationship between the degree of depolarization (and possibly Ca 2" entry) and AHP duration in the completely adapting cells. The relationship between AHP amplitude and spike area varied in a similar manner. Adult and repetitive firing immature neurons showed a slight increase in AHP amplitude with increased spike area, while AHP amplitude was largest with smaller areas in completely adapting immature cells. These relationships were unchanged by normalization of AHP amplitude by input resistance (Fig. 4D), again emphasizing that the larger and longer AHPs in completely adapting cells were not primarily due to differences in input resistance and spike width between immature and adult neurons. The remainder of the paper will concentrate on the sAHP, which exhibits the greatest postnatal changes. The prolonged sAHP in immature neurons appeared to be due to a K + current as suggested by the reversal potential ( - 1 0 9 mV; n = 2; derived by extrapolation from the line of best fit in a plot of AHP amplitude as a function of membrane potential). Assuming an intracellular (K +) of 120 mM t~, E K was estimated at - 9 8 mV using the Nernst equation. Replacement of extra-
CTL
J
L
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CO
co
-
-
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J
D -71 mV-
.~
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CTL
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5 mV ~ . [ 80O m
Fig. 5. T h e distinct firing pattern and prolonged A H P of completely adapting immature cells are Ca-dependent and apamin,insensitiv¢. A,B: a P15 neuron that did not fire throughout a 500 ms current injection in control aCSF, fired throughout the stimulus in zero Ca2+/2m M Co 2+ containing external solution. Repetitive firing was evoked with the same intensity stimulus (0.6 nA) in A and B. Spike amplitudes clipped d u e to digitization. C: the prolonged A H P was reduced when extracellular Ca 2+ was replaced with Co 2+ (2 mM). A H P s were evoked with 30, 2 ms current injections at 100 Hz (same cell as in A,B). D: the m A H P , but not the prolonged s A H P in a P9 neocortical cell was reduced by apamin (10 nM). A H P s were elicited by 20 repetitive 2 ms stimuli at 100 Hz.
219 cellular Ca 2+ with Co 2+ reduced the prolonged sAHP, suggesting that it was Ca-dependent (4 cells; Fig. 5C). When the prolonged AHP was blocked in zero Ca2+/2 mM Co2+-containing media, the cells fired repetitively throughout the stimulus (Fig. 5A,B). This suggests that a Ca-dependent K + conductance is largely responsible for spikeJrequency adaptation in immature as well as adult neurons. The sAHP was not blocked by apamin (a polypeptide from bee venom which in other cell types blocks AHP components 2~'34 and a particular class of Ca-dependent K + channel (SK)3); however, the mAHP was apamin-sensitive (10-100 nM; n = 4 / 4 ; Fig. 5D). We conclude that the relatively greater importance of Ca-dependent K + conductances underlying the sAHP in immature neocortical neurons results in altered firing patterns.
'tl
70 mV
A2
CONTROL
A/
Slow AHPs are modulated by transmitters (e.g., serotonergic, noradrenergic and muscarinic transmitters) in several adult cortical neurons ''s1~'9'-'3. We found that in adult rat neocortical neurons, NE (50-100 ~xM; six out of seven cells tested) decreased the sAHP, increased firing rate and f-I slope and decreased spike-frequency adaptation (Fig. 6). There was also a small reduction of peak mAHP amplitude with NE. The relatively greater expression of the Ca-dependent sAHP in immature neurons provides a different substrate for modulation than in adult neurons. We observed that NE had a proportionately greater effect in immature neurons due to the increased relative importance of the Ca-dependent sAHP (Fig. 7, compared to Fig. 6). In immature cells which fired repetitively throughout the stimulus, NE (25-100 txM) de-
NE
-70 m V ~
= :
-5B mv
- 0 mV~
NE
-58 mv
40 mV
i
40 SO
/
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~
30
480 640
E
130
70
10
40
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o CTL [] NE
70 60 50 40
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80
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20 200
400 TIME
600 (ms)
800
1000
200
400
600
TIME
(ms)
800
1000
Fig. 6. In adult rat neocortical neurons, N E (50/~M) affected the AHP, firing rate and spike-frequency adaptation. A: after bath application of NE, firing frequency was reversibly increased and spike frequency adaptation decreased. Spike amplitudes clipped due to digitization. Repetitive firing was evoked with the same intensity current injection (0.4 nA) in A I - A 3 . B: N E decreased the sAHPs (10 spikes elicited by 2 ms current injections at 100 Hz). C: steady-state firing frequency (average of last 500 ms of firing) plotted vs. injected current intensity. NE increased the rate of firing (control f-1 slope = 48.9 H z / n A , N E = 81.8 H z / n A ) . D: instantaneous firing frequency plotted vs. time during a l-s firing episode. To compare the adaptation in control and NE, stimulus intensity (0.5 nA) is matched. N E caused a modest decrease in spike-frequency adaptation. 12: final firing frequency is matched to accentuate the difference between N E and control. The y-axis has been clipped to expand the early difference in adaptation (CTL = 0.5 nA, N E = 0.3 hA).
220
A
B
CTL
ii 20 mV 2 nA
__ 320 ms
-67 m V ~
-67 mV
I
~ j
NE (loo ~M)
k
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WASH
C
D
CTL vs. NE vs. W A S H
-58 MV
II
\
.w,s. CTl_
-67 mV
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Fig. 7. NE altered firing behavior in immature neocortical neurons. A: a P21 cell would not fire throughout the stimulus pulse in control aCSF. B: after application of NE (100/zM), the cell could now fire regularly throughout the pulse. C: the effect of NE was partially reversible after washing in control aCSF (for 42 min.). Strong adaptation is apparent in the wash, but it is not as extreme as in control aCSF before NE. Repetitive firing was evoked with the same intensity stimulus (0.8 nA) in A - C . D: the prolonged sAHP was reduced in NE. This reduction was partially reversible upon wash in control aCSF. Same cell is shown in A - D .
creased the Ca-dependent sAHP, increased firing rate and decreased spike-frequency adaptation as in adult neurons (data not shown). In completely adapting immature cells, NE reduced the sAHP (Fig. 7D) and caused repetitive firing throughout the stimulus (Fig.
A
P6
!t 10 m V
B
1 I
i~
7A,B). This effect was reversible (Fig. 7C). This reduction of the sAHP by NE appeared qualitatively similar at different postnatal ages (29 of 35 cells tested;Fig. 8). In Fig. 8 it is also evident that the AHPs remaining after NE application is very similar at all ages, suggest-
p,
°"
C
P21
I
c"
1.6s
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r
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E
ADULT
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Fig. 8. NE reduced the sAHP at all ages during postnatal development. Because some immature cells had a large amplitude, prolonged s,M-tP, similar doses of NE had a proportionately greater effect at younger ages, AHPs were elicited by 30 spikes (2-ms current injections) at 100 Hz (firing not shown). Note similarity at all ages in A H P component remaining in NE.
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Fig. 9. The effects of NE on repetitive firing and AHPs are ~-mediated in immature cortical neurons. A: a completely adapting P15 neuron that would not fire throughout a 200 ms stimulus pulse (0.8 nA). Inset: isoproterenol decreased the prolonged AHP in this same cell. AHPs were elicited by 20 spikes at 100 Hz (2 ms stimulus pulses). Action potential amplitudes clipped due to digitization. B: the same cell fired throughout the same intensity stimulus in isoproterenol (100 # M ) C: isoproterenol decreased the sAHP and this effect was reversed upon return to control solutions. D: clonidine (100 ~M; a 2 agonist) applied to the same cell after wash had no effect on the AHP. AHPs in C and D elicited by 10 spikes at 100 Hz. E: NE decreased the sAHP in a different cell from A - D . After washing with timolol alone, the AHPs recovered. F: timolol (25 txM; 13-antagonist) applied together with NE blocked the effects of NE on the AHP. Same cell as in E. AHPs in E and F elicited by 20 spikes at 100 Hz.
ing the NE-sensitive portion of the AHP undergoes an age-dependent reduction during postnatal development. Since this same component is reduced by replacement of extracellular Ca 2+ with Co 2+ and reverses near the estimated EK, it is likely that the developmentally regulated sAHP is due to a Ca-dependent K + conductance. The effect of NE on the sAHP was mediated by /3-adrenergic receptors. The /3-agonist, isoproterenol (100 /-~M; n = 14/14), mimicked the actions of NE on repetitive firing and the sAHP at all ages (Fig. 9A-C). Clonidine (az-agonist; 100 p~M; n = 5 / 5 ; Fig. 9D) or phenylephrine (a~-agonist; 100 ~M; n = 3/3; data not shown) had no effect on the AHPs or firing behavior. The effects of NE on sAHPs were blocked or reduced by the /3-antagonist timolol (25 /_~M; n = 6/6; Fig. 9E, F).
A considerable amount of the morphological and physiological maturation in rat sensorimotor cortex occurs postnatally. Many electrical properties change during the first 4 postnatal weeks including action potential width, membrane time constant and input resistance (Kriegstein et al. 17, McCormick and Prince 22, present results). Changes also occur in repetitive firing behavior during this 4-week period. In particular, spike-frequency adaptation is much stronger in immature neurons. A principal finding of our study is that during the first postnatal week, the majority of presumed pyramidal cells will only fire for less than 300 ms regardless of the intensity of long depolarizing current injections (1 s). The proportion of cells with this complete adaptation decreases with age. Spikefrequency adaptation in immature neurons appears to be primarily controlled by Ca-dependent K + conductances as in mature neurons. Evidence for this comes from the Ca-dependence and NE-sensitivity of the sAHP and spike-frequency adaptation. The reversal potential of the sAHP suggests that it is K+-mediated. A previous study reported that AHPs were larger in immature rat neocortical neurons and that f-I slopes were less steep than in adult neurons (McCormick and Prince22). In that study, however, analysis was restricted to cells which fired repetitively, thus the completely adapting cells analyzed here were excluded. We have extended their observations on repetitive firing neurons, explicitly described the A H P components and have in addition documented that in immature neurons spike-frequency adaptation and the sAHP are mediated in large part by Ca-dependent K + currents. The firing behavior we have described for completely adapting immature cells appears quite different from that of adult neocortical neurons of several species and appears more similar to that of adult hippocampal CA1 neurons, which also fire for 200-300 ms at the beginning of a long stimulus pulse and thus exhibit complete spike-frequency adaptation (e.g., Madison and Nicolllg'2°). In CA1 neurons, spike-frequency adaptation (also referred to as accommodation) is produced mainly by a slow Ca-activated K + conductance, with an additional contribution from a voltage-gated K + current (M-current, Madison and Nicoll2°). Spikefrequency adaptation and the slow AHP in CAI neurons also are reduced by NE (Madison and Nicoll'~). The detailed mechanisms underlying the long duration and large amplitude of the sAHP in immature versus adult neocortical neurons remain to be determined. The prolonged sAHP in completely adapting immature cells is not primarily due to the higher input
222 resistance or b r o a d e r action potentials of i m m a t u r e cells. T h e reversal potential and the C a - d e p e n d e n c e of the s A H P suggests that it is not due to activation of a N a + - K * pump. Several other possibilities could explain this p r o l o n g e d A H P in i m m a t u r e cells. For example, C a - d e p e n d e n t K + c o n d u c t a n c e s in i m m a t u r e cells could have different properties than in m a t u r e cells. Alternatively, i m m a t u r e n e u r o n s could have relatively larger Ca 2+ currents thus leading to greater Ca 2+ availability. I m m a t u r e n e u r o n s may also handle Ca 2+ differently than adult cells. That is, for the same Ca z+ entry, there could be age-related differences in intracellular [Ca 2+] due to differences in Ca 2+ buffering, Ca 2+ exchange, or Ca 2+ release from internal stores. T h e different relationship we observed in completely adapting cells between A H P amplitude or duration and integrated spike area suggests that these cells may differ from adult neurons in the way depolarization (and presumably Ca z+ entry) is translated into the sAHP. A d u l t rat superior cervical ganglion cells and guinea pig vagal m o t o n e u r o n s have large, prolonged A H P s which a p p e a r grossly similar to the prolonged A H P s in i m m a t u r e rat neocortical n e u r o n s (Kawai and W a t a n a b e 16, Sah and McLachlan25). In these cell types, inhibiting Ca 2 + release from intracellular stores shortens the A H P and increasing Ca 2 + release e n h a n c e s the A H P (Kawai and W a t a n a b e 16, Sah and McLachlan25). Similar to the s A H P in neocortical neurons, the Ca-dep e n d e n t K + c o n d u c t a n c e underlying the prolonged A H P in vagal m o t o n e u r o n s is insensitive to apamin and r e d u c e d by NE. In vagal m o t o n e u r o n s , this conductance is decreased by agents which attenuate release from internal Ca 2+ stores (Sah and McLachlan25). A g e - r e l a t e d differences in firing behavior result in a different substrate for modulation. Inputs to developing rat neocortical n e u r o n s a p p e a r to be transduced differently than inputs to m a t u r e cells such that the faithfulness with which prolonged excitatory inputs are converted into action potentials would be diminished in i m m a t u r e cortical neurons relative to adult cells. T h e p r o l o n g e d s A H P may serve to reduce the t e n d e n c y for excitatory inputs to cause sustained depolarizations in i m m a t u r e cells which may not be able to handle large Ca z+ loads. Sustained depolarization and repetitive firing could lead to excessive Ca 2+ influx and p e r h a p s cell death (ChoiS). D u r i n g early postnatal development, the m a m malian neocortex is especially sensitive to changes in functional organization due to e x p e r i e n c e - d e p e n d e n t modifications (Rothblat et al. 24, Wiesel and Hubel3~). Several studies suggest that N E plays a role in ocular d o m i n a n c e plasticity in visual cortex (e.g. Bear and Singer 2, G r e u e l et al. 9, Kasamatsu and Pettigrew ~5) as
well as other forms of neuronal plasticity (for cxample, long-term potentiation in dentate gyrus and CA3 hipp o c a m p a l n e u r o n s : Bliss ct al. 4, H o p k i n s and Johnstonl2). Singer et al. 2v have proposed that a modification threshold involving Ca 2. influx through voltage-gated or N M D A - a c t i v a t e d Ca 2 ~ channels must be reached for such plastic changes to occur in the developing cortex. In this model, the probability of Ca 2+ entry is increased with correlated activity of pre- and post-synaptic neurons (sustained depolarization would o p e n voltage-gated channels or remove Mg 2÷ block of N M D A - a c t i v a t e d channels). Transmitters such as N E could thus affect neuronal plasticity at several levels: (1) increasing or correlating activity; (2) depolarizing the cell; (3) facilitating N M D A responses; (4) facilitating Ca 2+ entry directly t h r o u g h Ca 2+ channels; or (5) decreasing K + conductances, thus indirectly increasing Ca 2+ influx by either route (no. 3 or 4). We have provided evidence for a decrease in a specific Ca-dep e n d e n t K + c o n d u c t a n c e which underlies the s A H P and spike-frequency adaptation in immature neocortical pyramidal neurons. As the s A H P would tend to restrict the duration of Ca 2 + entry and thus Ca-related synaptic plasticity, N E ' s reduction of A H P conductances could indirectly regulate attainment of a modification threshold for developmental synaptic plasticity. Acknowledgements. We thank W. Armstrong and D.J. Surmeier for
valuable comments on an earlier version of this manuscript. This work was supported by National Institute of Neurological Disorders and Stroke Grant NS-27180 (to R.C.F.).
REFERENCES 1 Andrade, R. and Nicoll, R.A., Pharmacologically distinct actions of serotonin on single pyramidal neurones of the rat hippocampus recorded in vitro, J. Physiol., 394 (1987) 99-124. 2 Bear, M.F. and Singer, W., Modulation of visual cortical plasticity by acetylcholine and noradrenaline, Nature, 320 (1986) 172-176. 3 Blatz, A.L. and Magleby, K.L., Ca-activated potassium channels, Trends Neurosci., 10 (1987) 463-467. 4 Bliss, T.V.P., Goddard, G.V. and Riives, M., Reduction of longterm potentiation in the dentate gyrus of the rat following selective depletion of monoamines, J. Physiol., 334 (1983) 475-491. 5 Choi, D.W., Ca-mediated neurotoxicity: relationship to specific channel types and role in ischemic damage, Trends Neurosci.. 11 (1988) 465-469. 6 Connors, B.W., Gutnick, M.J. and Prince, D.A., Electrophysiological properties of neocortical neurons in vitro, J. Neurophysiol., 48 (1982) 1302-1320. 7 Foehring, R.C., Lorenzon, N.M., Herron, P. and Wilson, C.J., Correlation of physiologicallyand morphologically identified neuronal types in human association cortex in vitro, .,( Neurophysiol., 66 (1991) 1825-1837. 8 Foehring, R.C., Schwindt, P.C. and Crill, W.E., Norepinephrine selectively reduces slow Ca 2+- and Na+-mediated K + currents in cat neocortical neurons, J. Neurophysiol., 61 (1989) 245-256. 9 Greuel, J.M., Luhmann, H.J. and Singer, W., Pharmacological induction of use-dependent receptive field modifications in the visual cortex, Science, 242 (1988) 74-77.
223 10 Hamill, O.P., Huguenard, J.R. and Prince, D.A., Patch clamp studies of voltage gated currents in identified neurons of rat cerebral cortex, Cerebral Cortex, 1 (1991) 1-6. 11 Harvey, J.A., Schofield, C.N. and Brown, D.A., Evoked surfacepositive potentials in isolated mammalian olfactory cortex, Brain Res., 76 (1974) 235-245. 12 Hopkins, W.F. and Johnston, D., Frequency-dependent noradrenergic modulation of long-term potentiation in the hippocampus, Science, 226 (1984) 350-352. 13 Horikawa, K. and Armstrong, W.E., A versatile means of intracellular labelling: injection of biocytin and its detection with avidin conjugates, J. Neurosci. Methods, 25 (1988) 1 12. 14 Huguenard, J.R., Hamill, O.P. and Prince, D.A., Sodium channels in dendrites of rat cortical pyramidal neurons, Proc. Natl. Acad. Sci. USA, 86 (1989) 2473-2477. 15 Kasamatsu, T. and Pert)grew, J.D., Preservation of binocularity after monocular deprivation in the striate cortex of kittens treated with 6-hydroxydopamine, J. Comp. Neurol., 185 (1979) 139 162. 16 Kawai, T. and Watanabe, M., Effects of ryanodine on the spike after-hyperpolarization in sympathetic neurones of the rat superior cervical ganglion, Pfliigers Arch., 413 (1989) 47(I 475. 17 Kriegstein, A.R., Suppes, T. and Prince, D.A., Cellular and synaptic physiology and epileptogenesis of developing rat neocortical neurons in vitro, Dec. Brain Res., 34 (1987) 161-171. 18 Lorenzon, N.M. and Foehring, R.C., Relationship between repetitive firing and afterhyperpolarizations in human neocortical neurons, J. NeurophysioL, 67 (1992) 350-363. 19 Madison, D.V. and Nicoll, R.A., Noradrenaline blocks accomodation of pyramidal cell discharge in the hippocampus, Nature, 299 (1982) 636-638. 20 Madison, D.V. and Nicoll, R.A., Control of the repetitive discharge of rat CA1 pyramidal neurones in vitro, J. Physiol., 354 (1984) 319-331. 21 McCormick, D.A., Connors, B.W., Lighthall, J.W. and Prince, D.A., Comparative electrophysiology of pyramidal and sparsely spiny stellate neurons of the neoeortex, J. Neurophysiol., 54 (1985) 782-806. 22 McCormick, D.A. and Prince, D.A., Post-natal development of eleetrophysiological properties of rat cerebral pyramidal neurones, .1. Physiol., 393 (1987) 743-762.
23 Nowicky, A.V., Berry, R.L. and Teyler, T.J., Beta-adrenergic effects of norepinephrine studied in the rat visual cortical slice, Soc. Neurosci. Abstr., 15 (1989) 1058. 24 Rothblat, L.A., Schwartz, M.L. and Kasdan, P.M., Monocular deprivation in the rat: evidence for an age-related defect in visual behavior, Brain Res., 158 (1978) 456 460. 25 Sah, P. and McLachlan, E.M., Ca ~ +-act)wired K ~ currents underlying the afterhyperpolarization in guinea pig vagal neurons: a role for Ca2+-activated Ca z+- release, Neuron, 7 (1991) 257 264. 26 Schwindt, P.C., Spain, W.J., Foehring, R.C., Stafstrom, C.E., Chubb, M.C. and Crill. W.E., Multiple potassium conductances and their functions in neurons from cat sensor)motor cortex in vitro, J. Neurophysiol., 59 (1988) 424-449. 27 Singer, W., Tretter, F. and Yinon, U., Central gating of developmental plasticity in kitten visual cortex, .1. Physiol., 324 (1982) 221-237. 28 Szente, M.B., Baranyi, A. and Woody, C.D.. lntracellular injection of apamin reduces a slow potassium current mediating afterhyperpolarizations and 1PSPs in neocortical neurons of cats, Brain Res., 461 (1988)64-74. 29 Thompson, S.M., Masukawa, L.M. and Prince, D.A., Temperature dependence of intrinsic membrane properties and synaptic potentials in hippocampal CAI neurons in vitro, J. Neurosci.. 5 (1985) 817-824. 30 Uylings, H.B.M., Van Eden. C.G., Parnave!as, ,I.G. and Kalsbeck, A., The prenatal and postnatal development of rat cerebral cortex. In B. Kolb and R.C. Tees (Eds.), Cerehral ('ortex (~f the Rat,MIT Press, Cambridge, MA, 199(I, pp.35--76. 31 Wiesel, T.N. and Hubel, D.lq.. The period of susceptibility to lhe physiological effects of unilateral eye closure in kittens, J. Physiol., 206 (197(I) 419-436. 32 Wise, S.P. and Jones, E.G.. The organization and postnatal development of the commissural projection of the rat somatic sensory cortex, J. Comp. Neurol., 168 (1976) 313 344. 33 Wise, S.P. and Jones, E.G., Developmental studies of thalamocortical and commissural connections in the rat somatic sensory cortex, J. Ck>rnp. Neurol., 178 (1978) 187-208. 34 Zhang, L. and Krnjevic, K., Apamin depresses selectively the after-hyperpolarization of cat spinal motoneurons, Neurosci. Left., 74 (1987) 58-62.
213
BRESD 51635
The ontogeny of repetitive firing and its modulation by norepinephrine in rat neocortical neurons N.M. Lorenzon and R.C. Foehring Department of Anatomy and Neurobiology, The Unit'ersity of Tennessee at Memphis. Memphis, TN 38103-4~)01 ( UX4 (Accepted 22 December 1992)
Key words." Cortex; Development; Afterhyperpolarization; Norepinephrine: Repetitive firing: Modulation
The postnatal ontogeny of electrical properties was studied in rat sensorimotor cortical neurons (P6 to adult) using intraccllular recording in a n in vitro slice preparation. Many action potential properties and input resistance changed during the first 4 postnatal weeks. Repetitive firing behavior also changed during the first postnatal month. Spike-frequency adaptation was much stronger in immature neurons. At 1 week postnatal, the majority of cortical neurons would only fire for less than 200 ms regardless of the intensity of long depolarizing current injections. These cells were normal in other parameters and could fire throughout a depolarizing current injection in the presence of inorganic calcium channel blockers or norepinephrine (NE), suggesting that the inability to fire was not due to injury. The frequency with which we encountered cells with this extreme adaptation decreased with age. Spike-frequency adaptation in immature neurons appears to be primarily controlled by Ca-dependent K + conductances as in mature neurons. In mature and immature neurons, three afterhyperpolarizations (AHPs) could be distinguished by their rate of decline. The fast AHP followed repolarization of a single spike and was only partially Ca- and K-dependent. The medium duration AHP was Ca-dependent and apamin-sensitive and the slow AHP was partially Ca-dependent and not blocked by apamin. NE decreased the slow Ca-dependent AHP via /3-adrenergic receptors. This effect of NE on AHPs appeared qualitatively similar throughoul postnatal development. NE had a proportionately greater effect in younger neurons, however, due to their relatively larger slow AHP. The quantitative differences of NE's action on the slow AHP (sAHP) led to a qualitative difference in NE's effect on firing behavior. The effects of NE on firing behavior may therefore be greater during times critical for cortical maturation.
INTRODUCTION
c h a n g e d u r i n g the first p o st n at al m o n t h . Projections of t h a l a m o c o r t i c a l and callosal a f f e r e n t s c o n t i n u e to de-
A t birth, n e u r o n s
in rat s e n s o r i m o t o r c o r t e x are
v el o p postnatally 32'~3. S y n a p t o g e n e s i s c o n t i n u e s until
m o r p h o l o g i c a l l y an d electrically i m m a t u r e and several
a b o u t P28 3° In i m m a t u r e n e u r o n s , electrically e v o k e d
c h a n g e s o c c u r in the electrical p r o p e r t i e s
of t h e s e
postsynaptic p o t e n t i a l s (PSPs) are l o n g e r in latency and
n e u r o n s over the first 4 p o s t n a t a l weeks. A c t i o n p o t e n -
d u r a t i o n and are m o r e subject to failure during repeti-
tials are q u i t e b r o a d at birth, but th e i r width d e c r e a s e s
tive s t i m u l a t i o n 17. T h e relative b al an ce of synaptic exci-
t h r o u g h p o s t n a t a l wee k s 1 - 4 (the rates o f b o t h rise and
tation and inhibition also c h a n g e s with age. F o r exam-
r e p o l a r i z a t i o n increase), with the most d r a m a t i c c h a n g e s o c c u r r i n g d u r i n g the first p o s t n a t a l w e e k 17'22.
ple, d u r i n g the first p o st n at al week, synaptic inhibition is not o b s e r v e d in rat n e o c o r t e x iT.
T h e m a x i m u m rate o f rise o f Ca 2+ spikes also in-
T h e s e p o s t n a t a l m o r p h o l o g i c and synaptic c h a n g e s
c r e a s e s t h r o u g h o u t this t i m e 22. T h e s e c h a n g e s in action
parallel o n t o g e n e t i c c h a n g e s in the integrative ability
p o t e n t i a l s are t h o u g h t to r e f l e c t i n c r e a s e s in density o f
of cortical n e u r o n s .
Na +, K + and Ca 2+ c h a n n e l s d u r i n g p o s t n a t a l d e v e l o p -
r e l a t i o n s h i p b e t w e e n f r e q u e n c y and injected c u r r e n t
m e n t (c.f. refs. 10,14). I n p u t resistance, specific m e m -
(f-l)
b r a n e r e s i s t a n c e an d m e m b r a n e t i m e c o n s t a n t all dec r e a s e o v e r this 4 - w e e k p e r i o d 17'22.
a v e r a g e d u r a t i o n o f the slow a f t e r h y p e r p o l a r i z a t i o n s ( A H P s ) which follow r e p e t i t i v e firing is longer, in thc i m m a t u r e n e u r o n s 22. In adult cortical n e u r o n s ~'1~'21"2~',
T h e a f f e r e n t inputs to t h e s e cortical n e u r o n s also
has b e e n
F o r e x a m p l e , the slope of the
reported
to be less s t e e p
and
the
Correspondence: N.M. Lorenzon, Department of Anatomy and Neurobiology, 855 Monroe Avenue, The University of Tennessee at Memphis, Memphis, TN 38103-4901, USA. Fax: (1) (901) 528-7193.
214 t h e A H P s e l i c i t e d by s p i k e t r a i n s r e f l e c t c o n d u c t a n c e s
W e test t h e h y p o t h e s i s t h a t a q u a l i t a t i v e c h a n g e in the
a c t i v a t e d by t h e s u s t a i n e d d e p o l a r i z a t i o n d u r i n g firing.
m o d u l a t i o n by N E o c c u r s d u r i n g d e v e l o p m e n t .
T h e s l o w e r A H P s a r e d o m i n a t e d by C a - d e p e n d e n t
K ~ MATERIALS AND METHODS
c o n d u c t a n c e s w h i c h a r e p r i m a r i l y r e s p o n s i b l e for s p i k e frequency
adaptation <~s'2~'>. M u c h
less is k n o w n , h o w -
For the in vitro slice preparation, a block of tissue was removed from the sensorimotor cortical region of pentobarbital sodium (35 mg/kg) or metofane anesthetized rats (n = 61). The tissue sample was immediately sectioned at 400-500 p.m (for the first 2 postnatal weeks thicker sections were used; we have found this increases cell viability) in artificial cerebrospinal fluid (aCSF), containing (in mM): 125 NaCI. 3 KCI, 2 CaCI 2, 5 MgCI2, 1.2 NaH2PO4, 26 NaHCO 3, 20 dextrose, (pH = 7.4; 310-320 mOsm/I). Slices were cut at 4°C using a WPI oscillating tissue slicer. Slices were then incubated in holding chambers containing aCSF bubbled with carbogen (95% O~/5% CO 2) at 30°C for at least 1 h. Individual slices were then transferred to an interface recording chamber maintained at 34°C (tissue from immature rats was held at 34°C to improve viability ~7. Slices from adult rats were also kept at this temperature to control tk~r the temperature-dependence of AHPs>). The slice was bathed in oxygenated aCSF which flowed under the slice. Carbogen-saturated water vapor flowed over the top of the slice. To observe the effects of zero Ca :+ on cells, the Ca z+ in the aCSF was replaced with 2 mM Co 2+ and the NaH2PO 4 was omitted to avoid precipitation. Apamin (10-200 nM), NE and adrenergic agonists and antagonists were added directly to the aCSF: arterenol-bitartrate salt (25-100 /zM), isoproterenol-HCl (50-100 /xM), clonidine-HCI (100 /xM), phenylephrine-HCI (100 /xM), timolol-maleate salt (25 /xM). All drugs were purchased from Sigma except isoproterenot and clonidine (RBI). Microelectrodes (40-180 M~fl) were filled with 2 M potassium acetate, 2 M KMeSO 4, or 1% biocytin/2 M potassium acetate solutions. Tissue sections containing cells filled with bioeytin were processed histochemically using the ABC technique (modified from ref. 13; see ref. 7). All cells that were successfully filled and recovered were pyramidal cells (n = 19). No fast-spiking or intrinsic burstfiring neurons zt were included in this study. Recordings were from layers II-V (with most in layer II/1II or V). All recordings utilized an Axoclamp-llb electrometer (Axon Instruments) in continuous bridge or discontinuous current clamp
e v e r , a b o u t t h e s e p r o c e s s e s in i m m a t u r e c o r t i c a l n e u rons. In s e v e r a l a d u l t s p e c i e s , n o r e p i n e p h r i n e
(NE)
af-
fects t h e A H P s a n d firing b e h a v i o r o f c o r t i c a l n e u r o n s . F o r e x a m p l e , in a d u l t cat B e t z cells, N E r e d u c e d t h e slow A H P , i n c r e a s e d firing r a t e a n d d e c r e a s e d spikef r e q u e n c y a d a p t a t i o n s. T h e s e a c t i o n s o f N E w e r e p r o d u c e d by r e d u c t i o n o f a slow C a - d e p e n d e n t
K + con-
d u c t a n c e via /3~ r e c e p t o r s s. N E has b e e n i m p l i c a t e d as influencing ocular dominance
plasticity in d e v e l o p i n g
v i s u a l c o r t e x (e.g. refs. 2,9,15). S i n g e r a n d c o l l e a g u e s 27 h a v e p r o p o s e d t h a t for t h e s e c h a n g e s to o c c u r , a m o d i fication threshold must be reached. If NE blocked a Ca-dependent
K + conductance
in i m m a t u r e
cortical
n e u r o n s as in a d u l t n e u r o n s , t h e i n h i b i t o r y e f f e c t s o f this K + c u r r e n t w o u l d b e r e d u c e d a n d t h e p r o b a b i l i t y that
the
neuron
would
reach
such
a
modification
t h r e s h o l d w o u l d b e i n c r e a s e d . It is t h e r e f o r e i m p o r t a n t to u n d e r s t a n d
the mechanisms of action of NE during
p o s t n a t a l d e v e l o p m e n t , w h e n p l a s t i c c h a n g e s in c o r t i cal w i r i n g a r e m o s t e v i d e n t . In t h e p r e s e n t study, w e d e s c r i b e t h e o n t o g e n y o f r e p e t i t i v e firing b e h a v i o r in n e o c o r t i c a l p y r a m i d a l n e u rons and initiate studies concerning the development of modulation
of AHPs
a n d r e p e t i t i v e firing by N E .
P8
A1
P15
P29
P22
B1
C1
,.mv
D1 -72 mV -~
I
80 mV 4nA l ~ 8 ms
A2 -62 mV
-68
-78 mV
240 ms
Fig. 1. Changes in electrical properties during the first 4 postnatal weeks. A1-DI: spike width decreased with age. Action potentials elicited with 5 ms stimulus pulses. A2-D2: rheobase and input resistance traces for the first 4 postnatal weeks. Input resistance decreased, but rheobase did not change throughout postnatal development.
215 TABLE I
Electrical properties during postnatal det'elopment Values expressed as m e a n + S . D . Spike width measured at half amplitude. Rheobase is the minimum amount of current to elicit an action potential with 200 ms current injection. * indicates properties which showed significant differences between age groups in an analysis of variance test (~ = (l.05). Other symbols note significant differences with pairwise comparison using post hoc test: ~, different from P6-10: ", different from PI2 17: #, different from P20-22; o different from P27-33: +, different from adult group. Number of animals for each age group: 10 (for 6-111 days), 18 (for 12-17 days), 6 (for 20-22 days), 5 (for 27 33 days), 22 (for adult).
Electrical
Age of rat
properties
6 - 10 days
12 - 17 days
20 - 22 days
2 7 - 33 days
aduh
Resting potential (mY)
66 + 7 (24) 76.3±8.1 (26) #©+ 1.93 ± 0.75 (24) ~ # o + 182+98 (17) #c> + 49+_ 19 (17) o 0.31 +0.33 (24) 87.11 ± 46.3 (18) ~#©+
- 67 ± 6 (81) 83.0+ 13.4 (71) #:: " 1.35 _+{).56 (72) ~#o+ 219+ 109 (59) < ~ 49+ 15 (59) # c 0.32+0.33 (70) 61.8 + 30.8 (60) ~
- 68 + 6 (32) 90.3+_ 14.3 (29) ~ 0.97 + I).28 (29) ~ 274±91 (25) + 71 + 16 25) 0.30±0.14 (25) 45.8 ± 18.1 (23) ~
68 + 6 (19) 97.3+ 10.4 (21) '~ ~ 0.70 + 0.14 (21) ~'" 351 + 117 (18) ~ = # 103+36 (18) ~'' # I).27+0.16 ( 171 50.3 + 37.2 (19) ~
-- 70 + 7 (47) ~9.8± 11.3 192} " " 0.77 + (}.22 (89) ~'~' 347+ 87 (80) ~ " '~ 73+40 (8111 ~ " (}.40 + 1t. 13 (83) 42.0 + 22.8 (74) ~:~
AP amplitude (mV) * Spike width (ms) *
d V / d t , up ( V / s ) * d V / d t , down ( V / s ) * Rheobase (nA) Input resistance (M 11) *
mode. Data were recorded and stored on videotape using a Neurocorder (Neurodata) and a VCR. A H P s and firing frequency were visualized and measured off-line with RC Electronics Computerscope. The resting membrane potentials were estimated from continuous chart recordings of DC potential. Continuous monitoring of m embr ane potentials on the chart recorder allowed estimates to be made regarding drift in cell membrane potential due to electrode polarization. All statistical data are presented as mean ± S.D. Statistical differences between various age-groups for electrical properties was determined by an analysis of variance ( a = 0.05 level). A post hoc test (Tukey's HSD) was used for those measures in which a significant difference was found in the analysis of variance.
RESULTS During the first 4 postnatal weeks, several changes in neuronal electrical properties of neocortical cells were observed. Action potential width (measured at half amplitude) and input resistance decreased with age of the animal. Action potential amplitudes and the rates of rise and repolarization of action potentials increased with age (Fig. 1, Table I; see also refs. 17,22). Resting membrane potential did not vary significantly with age. In response to prolonged (500-1000 ms) current injection, adult rat cortical neurons fire repetitively throughout the stimulus (Fig. 2A). The average rate of firing increases linearly or bilinearly with increased injected current (Fig. 2B, Table II). Instantaneous firing frequency (1/interspike interval) is initially high in adult cells, but decreases with time during the stimulus (spike frequency adaptation: f-t relationship, Fig. 2D). Some cells from 1-week-old animals also fired repetitively, as in adult neurons. In these cases, the slope of
the f-1 relationship was less steep (Table It, Fig. 2B) and spike-frequency adaptation was stronger than in the adult. T h e f-I slope increased with age (f-I slope at 1 week = 25.2_+ 7.4 H z / n A (n = 51, at 2 weeks = 35.5 _+ 15.2 H z / n A (n = 101, at 3 weeks = 37.6 + 13.3 H z / n A (n = 5), at 4 weeks = 46.0 _+ 14.0 H z / n A (n = 4)). Several cells from 1-3-week-old rats had a different firing pattern; regardless of the intensity of the injected current, these cells would only fire through the first 100-250 ms of a 1-s depolarizing current injection (Fig. 2C,D). This period of firing was followed by a pronounced, prolonged slow A H P (Fig. 3D,F). In some cases, firing would resume upon termination of this A H P . Even t h o u g h these cells only fired a few spikes at
T A B L E II
Differences in firing and AHf3~' in 1-week-old and adult neocortieal neurons Repetitive firing evoked by 20-2 ms repetitive stimuli at 100 Hz at resting me mbra ne potential. # Includes only l-week-old cells tha! would fire throughout a l-s current injection. All of the above pa ra me t e rs significantly differed between 1-week-old and adult neurons (Student's t-test; a = 11.05). Resting membrane potentials did not differ significantly between age groups.
1-week-old mAHP (after 1 spike; mV) m A H P (after repetitive firing, mV) sAHP(mV) s A H P duration (s) f-lslope(Hz/nA) #
4.5 ± 3.9 10.5 + 1.2 5.6 +1.3 4.26+0.82 25.2 +7.4
Adult (22) (8) (8) (8) (5)
3.0 ±
1.6 (20)
9.1 + 1.3 1.9 ± 0.6 1.49± 0.42 57.5 ±47,7
(7) (7) (7) (10)
216 ADULT
the beginning of a depolarizing pulse, the slow AHP (sAHP) was still greater in amplitude and duration than in adult cells which fired throughout the same stimulus. The frequency with which we encountered cells with this firing pattern (100% adaptation at 300 ms during a l-s current injection) decreased with age (Fig. 2D inset). We will refer to these cells as completely adapting cells. During the first postnatal week a majority of the cells (58%) were completely adapting; 37% of the cells from 2-week-old animals and 25% of the cells from 3-week-old rats also showed this firing pattern. We did not observe completely adapting cells
-73 mV
C
/r::::
-77 rnV=-J-- -
mAH~
ADULT
P15
I[
ADULT
E
40
B
'omvI
-
~
P15
HP
I I mAHP s
F
p
P9
30-
~
loo0.2
04
0.6
-75 mV
D ;~
_~o
tJ
<
t
Z
,,~
~
2 3 4 z5 A G E (weeks)
40
o -67 m V ~
(nA)
°o 160
ms
1.0
0.8
CURRENT
.v, ~"
,
200
•
TIME
,
•
,
400
,
•
,
600
(ms)
Fig. 2. Repetitive firing behavior in adult and 1-week-old rat neocortical neurons. AI: adult regular-spiking cell fires throughout a 1-s depolarizing stimulus pulse (0.3 nA) at resting membrane potential (RP). A2: increasing the stimulus current injection (0.8 nA) increases the rate of firing. Spike amplitude clipped due to digitization. B: steady-state firing frequency plotted vs. injected current intensity. The average frequency of firing during the last 500 ms of firing is shown for the adult cell illustrated in A1, 2 (open symbols) and a P15 neuron which fired repetitively (filled symbols, different from cell in C1 and C2). The solid line through the adult data is a least squares fit with slope of 47.6 Hz/nA. CI: repetitive firing in a P15 neocortical neuron. Some immature neurons would not fire repetitively throughout a long, depolarizing current injection (0.8 nA; 100% spike-frequency adaptation by 200 ms). A spike was evoked towards the end of the stimulus pulse (probably due to sufficient decay of the prolonged AHP). C2: increasing the stimulus pulse (1.2 hA) increases the number of spikes at the beginning of the pulse, but the cell still does not fire throughout the stimulus. D: adult (open symbols, same cell as in A) and P15 (closed symbols, same cell as in C) neocortical neurons exhibit spike-frequency adaptation (0.8 nA current injection). Instantaneous firing frequency (1/interspike interval) plotted vs. time during a 1-s repetitive firing episode. The young cell stops firing within the first 100 ms (100% adaptation). Inset: the frequency with which we encountered cells with this firing pattern (100% adaptation by 300 ms) decreased with age.
Fig. 3. AHPs in adult and immature neocortical neurons. A,B: AHPs evoked by a single spike in adult (A) and a P15 cortical celt(B). A fast AHP (fAHP) could be observed as a notch or interruption in spike repolarization and a medium AHP (mAHP) follows the delayed depolarization (DD). C,D: AHPs evoked by repetitive firing episode. The mAHP immediately follows the train of spikes. The slow AHP (sAHP) follows the mAHP. Note that the sAHP in the immature neuron is much larger and longer despite the shorter duration of firing. In the immature neuron, the mAHP is evident as a brief notch before the development of the sAHP. E,F: the AHPs increased in amplitude with increasing number of spikes: AHPs from an adult neuron (E) elicited by repetitive 2 ms stimuli at 100 Elz (at resting potential (RP); 5, 10, and 30 spikes). The sAHP is relatively short and l0 spikes or more are required in order to elicit a measurable sAHP. AHPs from a P9 cell (F) were elicited by repetitive 2-ms stimuli at 100 Hz (at RP; 2, 5, 10 and 30 spikes). A sAHP is measurable after only 2 spikes and after 5 spikes appears kinetically distinct from the faster AHPs. A, C and E are all from the same cell and B and D are from another cell.
from rats older than P28. The remaining cells fired repetitively. The inability to fire repetitively throughout a current injection could indicate that the celt was injured by the impalement. Since immature neurons are typically more difficult to record from, it is important to distinguish between injured neurons and a distinct firing pattern. We only considered cells in which 100% adaptation was accompanied by a large AHP. In addition, immature cells with input resistances less than 20 MS2 or action potential amplitude less than 6 0 mV were not considered. The immature neurons in this study appeared healthy with respect to standard measures (action potential amplitude, input resistance and resting membrane potential). There were no differ-
217 pendent (data not shown; see also refs. 26,18) and increases in amplitude with multiple action potentials (Fig. 3C,E). Following a repetitive firing episode in adult rat neocortical cells, two AHPs with different rates of decay were present: a medium AHP (mAHP) and a slow AHP (sAHP). The mAHP was present after one spike (lasting 126.7 ms_+76.9 ms; n = 76) and increased in amplitude with additional spikes. Medium AHP amplitudes were measured as the peak hyperpolarization immediately following the firing episode (Fig. 3C,E, Table II). The sAHP duration decayed more slowly (over seconds), required 10-20 spikes to be elicited and was smaller in amplitude than the mAHP (Fig. 3C,E, Table II). The sAHP amplitude was measured 500 ms after cessation of the stimulus pulse (when decay of the m A H P should be nearly complete). These AHPs were similar to those previously reported in other neocortical n e u r o n s 6"18'26. To control for differences between cells from various ages in rate of firing, we elicited AHPs by evoking a set number of spikes at a constant frequency using repetitive 2-ms suprathreshold current injections. Whereas 10-20 spikes at 100 Hz were required to obtain a measurable sAHP in adult neurons, only 2-5 spikes were required in most immature neurons (Fig. 3E vs. 3F). This suggests that in younger animals the
ences in measured electrical parameters (action potential amplitude and width, input resistance, or rheobase) between those immature neurons which fired repetitively and those which were completely adapting. Stronger evidence for the health of these cells comes from the observation that these cells will fire throughout a long depolarizing stimulus pulse in the presence of inorganic Ca 2+ channel blockers (Fig. 5) or neurotransmitters (Fig. 7, see text below). The transmitter effects, in particular, are reversible (Fig. 7). Together these observations suggest that some neocortical neurons do not fire repetitively because, compared to adult cells they display a relatively greater expression of conductances underlying the sAHP. In cortical neurons of all ages, a single action potential is followed by a multiphasic afterpotential (Fig. 3A). Typically spike repolarization leads to an initial notch or pause in repolarization depending upon membrane potential. In cat 26 or human 18 neocortical neurons, this has been referred to as the fast A H P (fAHP) and is partially due to K + conductances. The fAHP is also seen as a sharp hyperpolarization during the interspike interval (ISI) during repetitive firing (Fig. 3C). After a single spike, the fAHP is followed by a delayed depolarization (DD) followed by a longer-lasting medium AHP (mAHP) or a depolarization followed by the mAHP (Fig. 3A, Table lI). This m A H P is Ca-de-
A
~
10Z
A
8
O
A
8-
I--
A
.J
~
B
A
~12
4
•
A
RA '~
0
• "o, " , 50
o .....
100
150
200
0
250
.:.!:. 7:. "., 4
8
12
AREA (mV-s)
INPUT RESISTANCE(MQ)
11 ADULT I O 1 WEEK OLD (REPETITIVELY FIRING) •1 WEEK OLD (COMPLETELY ADAPTING)
C
D D
31o z
(2_
8
•~
6¸
~ ZX ~
Q. "r
2
A A
A
:~ 0.20 ta~ ~ 0.,~
~
A
A
°/~o° o A o° AREA (mY-s)
o
0,I0
zx
A A A
z
o3
,5 0.oo
A Q
1'2
AREA (mY-s)
Fig. 4. Possible factors contributing to differences in the sAHP. A: s A H P amplitude is plotted as a function of input resistance of the cell. In all parts of this figure, open circles represent 1-week-old cells which fired repetitively, open triangles represent completely adapting 1-week-old cells and filled in squares represent adult cells. Cells with similar input resistance can have very different A H P amplitudes. B: A H P duration is correlated with the integrated area under the spikes in adult neurons (r = 0.79). C: the relationship between the A H P duration and integrated spike area differs between repetitive firing and completely adapting 1-week-old neurons. D: the relationship between A H P a m p l i t u d e / i n p u t resistance and the integrated area under the spikes in adult cells, repetitive firing immature cells and completely adapting immature cells.
218 conductances underlying the sAHP would more frequently play a role in regulating firing under physiological conditions. Since action potentials are broader and input resistance higher in immature neurons (Fig. 1, Table I), the longer A H P in immature cells could reflect a greater influx of Ca 2+ during the broader spike, with increased input resistance leading to a greater membrane voltage change in response to similar underlying A H P currents. The relative A H P amplitude was similar for adult and repetitive firing immature cells (Fig. 4A), but the A H P amplitude was consistently higher for completely adapting cells than for adult or repetitive firing immature cells at a given input resistance (Fig. 4A). Input resistance did not differ between repetitive firing and completely adapting immature neurons. To control for age-related differences in spike width, we integrated the area under the action potentials used to elicit AHPs for eight adult cells and ten neurons from 1-week-old rats (number of spikes was controlled by using a 2-ms repetitive stimuli train and 2 - 6 different spike trains with various numbers of spikes were analyzed for each cell). The area under the spikes correlated with the number of spikes in the train in both adult (r = 0.79 (20 cells)) and immature cells (r = 0.50 (31 cells)). In adult neurons, the duration of the sAHP increased with an increase in the area under the spikes (Fig. 4B). In the 1-week-old neurons, the AHP duration and area appeared to have a different
relationship than that in the adult (Fig. 4C). Repetitive firing cells from 1-week-old rats show a weak increase in AHP duration with area as in adult neurons (Fig. 4C). In contrast, completely adapting cells show all inverse relationship with the longest AHPs generated by smaller spike areas (smaller than 5 mV-s; Fig. 4C). These data suggest a different relationship between the degree of depolarization (and possibly Ca 2" entry) and AHP duration in the completely adapting cells. The relationship between AHP amplitude and spike area varied in a similar manner. Adult and repetitive firing immature neurons showed a slight increase in AHP amplitude with increased spike area, while AHP amplitude was largest with smaller areas in completely adapting immature cells. These relationships were unchanged by normalization of AHP amplitude by input resistance (Fig. 4D), again emphasizing that the larger and longer AHPs in completely adapting cells were not primarily due to differences in input resistance and spike width between immature and adult neurons. The remainder of the paper will concentrate on the sAHP, which exhibits the greatest postnatal changes. The prolonged sAHP in immature neurons appeared to be due to a K + current as suggested by the reversal potential ( - 1 0 9 mV; n = 2; derived by extrapolation from the line of best fit in a plot of AHP amplitude as a function of membrane potential). Assuming an intracellular (K +) of 120 mM t~, E K was estimated at - 9 8 mV using the Nernst equation. Replacement of extra-
CTL
J
L
C -59 m Y - " I ' - -
CO
co
-
-
I _ _
J
D -71 mV-
.~
!
CTL
CTL
5 mV ~ . [ 80O m
Fig. 5. T h e distinct firing pattern and prolonged A H P of completely adapting immature cells are Ca-dependent and apamin,insensitiv¢. A,B: a P15 neuron that did not fire throughout a 500 ms current injection in control aCSF, fired throughout the stimulus in zero Ca2+/2m M Co 2+ containing external solution. Repetitive firing was evoked with the same intensity stimulus (0.6 nA) in A and B. Spike amplitudes clipped d u e to digitization. C: the prolonged A H P was reduced when extracellular Ca 2+ was replaced with Co 2+ (2 mM). A H P s were evoked with 30, 2 ms current injections at 100 Hz (same cell as in A,B). D: the m A H P , but not the prolonged s A H P in a P9 neocortical cell was reduced by apamin (10 nM). A H P s were elicited by 20 repetitive 2 ms stimuli at 100 Hz.
219 cellular Ca 2+ with Co 2+ reduced the prolonged sAHP, suggesting that it was Ca-dependent (4 cells; Fig. 5C). When the prolonged AHP was blocked in zero Ca2+/2 mM Co2+-containing media, the cells fired repetitively throughout the stimulus (Fig. 5A,B). This suggests that a Ca-dependent K + conductance is largely responsible for spikeJrequency adaptation in immature as well as adult neurons. The sAHP was not blocked by apamin (a polypeptide from bee venom which in other cell types blocks AHP components 2~'34 and a particular class of Ca-dependent K + channel (SK)3); however, the mAHP was apamin-sensitive (10-100 nM; n = 4 / 4 ; Fig. 5D). We conclude that the relatively greater importance of Ca-dependent K + conductances underlying the sAHP in immature neocortical neurons results in altered firing patterns.
'tl
70 mV
A2
CONTROL
A/
Slow AHPs are modulated by transmitters (e.g., serotonergic, noradrenergic and muscarinic transmitters) in several adult cortical neurons ''s1~'9'-'3. We found that in adult rat neocortical neurons, NE (50-100 ~xM; six out of seven cells tested) decreased the sAHP, increased firing rate and f-I slope and decreased spike-frequency adaptation (Fig. 6). There was also a small reduction of peak mAHP amplitude with NE. The relatively greater expression of the Ca-dependent sAHP in immature neurons provides a different substrate for modulation than in adult neurons. We observed that NE had a proportionately greater effect in immature neurons due to the increased relative importance of the Ca-dependent sAHP (Fig. 7, compared to Fig. 6). In immature cells which fired repetitively throughout the stimulus, NE (25-100 txM) de-
NE
-70 m V ~
= :
-5B mv
- 0 mV~
NE
-58 mv
40 mV
i
40 SO
/
WASH
Dil
~
~
30
480 640
E
130
70
10
40
o13oi,o;5o16
CURRENT
(nA)
ms ms
o CTL [] NE
70 60 50 40
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80
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100
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20 200
400 TIME
600 (ms)
800
1000
200
400
600
TIME
(ms)
800
1000
Fig. 6. In adult rat neocortical neurons, N E (50/~M) affected the AHP, firing rate and spike-frequency adaptation. A: after bath application of NE, firing frequency was reversibly increased and spike frequency adaptation decreased. Spike amplitudes clipped due to digitization. Repetitive firing was evoked with the same intensity current injection (0.4 nA) in A I - A 3 . B: N E decreased the sAHPs (10 spikes elicited by 2 ms current injections at 100 Hz). C: steady-state firing frequency (average of last 500 ms of firing) plotted vs. injected current intensity. NE increased the rate of firing (control f-1 slope = 48.9 H z / n A , N E = 81.8 H z / n A ) . D: instantaneous firing frequency plotted vs. time during a l-s firing episode. To compare the adaptation in control and NE, stimulus intensity (0.5 nA) is matched. N E caused a modest decrease in spike-frequency adaptation. 12: final firing frequency is matched to accentuate the difference between N E and control. The y-axis has been clipped to expand the early difference in adaptation (CTL = 0.5 nA, N E = 0.3 hA).
220
A
B
CTL
ii 20 mV 2 nA
__ 320 ms
-67 m V ~
-67 mV
I
~ j
NE (loo ~M)
k
__J
I.__
WASH
C
D
CTL vs. NE vs. W A S H
-58 MV
II
\
.w,s. CTl_
-67 mV
.my
l~ 8~Oms
__r
1__
Fig. 7. NE altered firing behavior in immature neocortical neurons. A: a P21 cell would not fire throughout the stimulus pulse in control aCSF. B: after application of NE (100/zM), the cell could now fire regularly throughout the pulse. C: the effect of NE was partially reversible after washing in control aCSF (for 42 min.). Strong adaptation is apparent in the wash, but it is not as extreme as in control aCSF before NE. Repetitive firing was evoked with the same intensity stimulus (0.8 nA) in A - C . D: the prolonged sAHP was reduced in NE. This reduction was partially reversible upon wash in control aCSF. Same cell is shown in A - D .
creased the Ca-dependent sAHP, increased firing rate and decreased spike-frequency adaptation as in adult neurons (data not shown). In completely adapting immature cells, NE reduced the sAHP (Fig. 7D) and caused repetitive firing throughout the stimulus (Fig.
A
P6
!t 10 m V
B
1 I
i~
7A,B). This effect was reversible (Fig. 7C). This reduction of the sAHP by NE appeared qualitatively similar at different postnatal ages (29 of 35 cells tested;Fig. 8). In Fig. 8 it is also evident that the AHPs remaining after NE application is very similar at all ages, suggest-
p,
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C
P21
I
c"
1.6s
D
i1.
r
P29
E
ADULT
o°.
Fig. 8. NE reduced the sAHP at all ages during postnatal development. Because some immature cells had a large amplitude, prolonged s,M-tP, similar doses of NE had a proportionately greater effect at younger ages, AHPs were elicited by 30 spikes (2-ms current injections) at 100 Hz (firing not shown). Note similarity at all ages in A H P component remaining in NE.
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......
TIM ÷ NE
Fig. 9. The effects of NE on repetitive firing and AHPs are ~-mediated in immature cortical neurons. A: a completely adapting P15 neuron that would not fire throughout a 200 ms stimulus pulse (0.8 nA). Inset: isoproterenol decreased the prolonged AHP in this same cell. AHPs were elicited by 20 spikes at 100 Hz (2 ms stimulus pulses). Action potential amplitudes clipped due to digitization. B: the same cell fired throughout the same intensity stimulus in isoproterenol (100 # M ) C: isoproterenol decreased the sAHP and this effect was reversed upon return to control solutions. D: clonidine (100 ~M; a 2 agonist) applied to the same cell after wash had no effect on the AHP. AHPs in C and D elicited by 10 spikes at 100 Hz. E: NE decreased the sAHP in a different cell from A - D . After washing with timolol alone, the AHPs recovered. F: timolol (25 txM; 13-antagonist) applied together with NE blocked the effects of NE on the AHP. Same cell as in E. AHPs in E and F elicited by 20 spikes at 100 Hz.
ing the NE-sensitive portion of the AHP undergoes an age-dependent reduction during postnatal development. Since this same component is reduced by replacement of extracellular Ca 2+ with Co 2+ and reverses near the estimated EK, it is likely that the developmentally regulated sAHP is due to a Ca-dependent K + conductance. The effect of NE on the sAHP was mediated by /3-adrenergic receptors. The /3-agonist, isoproterenol (100 /-~M; n = 14/14), mimicked the actions of NE on repetitive firing and the sAHP at all ages (Fig. 9A-C). Clonidine (az-agonist; 100 p~M; n = 5 / 5 ; Fig. 9D) or phenylephrine (a~-agonist; 100 ~M; n = 3/3; data not shown) had no effect on the AHPs or firing behavior. The effects of NE on sAHPs were blocked or reduced by the /3-antagonist timolol (25 /_~M; n = 6/6; Fig. 9E, F).
A considerable amount of the morphological and physiological maturation in rat sensorimotor cortex occurs postnatally. Many electrical properties change during the first 4 postnatal weeks including action potential width, membrane time constant and input resistance (Kriegstein et al. 17, McCormick and Prince 22, present results). Changes also occur in repetitive firing behavior during this 4-week period. In particular, spike-frequency adaptation is much stronger in immature neurons. A principal finding of our study is that during the first postnatal week, the majority of presumed pyramidal cells will only fire for less than 300 ms regardless of the intensity of long depolarizing current injections (1 s). The proportion of cells with this complete adaptation decreases with age. Spikefrequency adaptation in immature neurons appears to be primarily controlled by Ca-dependent K + conductances as in mature neurons. Evidence for this comes from the Ca-dependence and NE-sensitivity of the sAHP and spike-frequency adaptation. The reversal potential of the sAHP suggests that it is K+-mediated. A previous study reported that AHPs were larger in immature rat neocortical neurons and that f-I slopes were less steep than in adult neurons (McCormick and Prince22). In that study, however, analysis was restricted to cells which fired repetitively, thus the completely adapting cells analyzed here were excluded. We have extended their observations on repetitive firing neurons, explicitly described the A H P components and have in addition documented that in immature neurons spike-frequency adaptation and the sAHP are mediated in large part by Ca-dependent K + currents. The firing behavior we have described for completely adapting immature cells appears quite different from that of adult neocortical neurons of several species and appears more similar to that of adult hippocampal CA1 neurons, which also fire for 200-300 ms at the beginning of a long stimulus pulse and thus exhibit complete spike-frequency adaptation (e.g., Madison and Nicolllg'2°). In CA1 neurons, spike-frequency adaptation (also referred to as accommodation) is produced mainly by a slow Ca-activated K + conductance, with an additional contribution from a voltage-gated K + current (M-current, Madison and Nicoll2°). Spikefrequency adaptation and the slow AHP in CAI neurons also are reduced by NE (Madison and Nicoll'~). The detailed mechanisms underlying the long duration and large amplitude of the sAHP in immature versus adult neocortical neurons remain to be determined. The prolonged sAHP in completely adapting immature cells is not primarily due to the higher input
222 resistance or b r o a d e r action potentials of i m m a t u r e cells. T h e reversal potential and the C a - d e p e n d e n c e of the s A H P suggests that it is not due to activation of a N a + - K * pump. Several other possibilities could explain this p r o l o n g e d A H P in i m m a t u r e cells. For example, C a - d e p e n d e n t K + c o n d u c t a n c e s in i m m a t u r e cells could have different properties than in m a t u r e cells. Alternatively, i m m a t u r e n e u r o n s could have relatively larger Ca 2+ currents thus leading to greater Ca 2+ availability. I m m a t u r e n e u r o n s may also handle Ca 2+ differently than adult cells. That is, for the same Ca z+ entry, there could be age-related differences in intracellular [Ca 2+] due to differences in Ca 2+ buffering, Ca 2+ exchange, or Ca 2+ release from internal stores. T h e different relationship we observed in completely adapting cells between A H P amplitude or duration and integrated spike area suggests that these cells may differ from adult neurons in the way depolarization (and presumably Ca z+ entry) is translated into the sAHP. A d u l t rat superior cervical ganglion cells and guinea pig vagal m o t o n e u r o n s have large, prolonged A H P s which a p p e a r grossly similar to the prolonged A H P s in i m m a t u r e rat neocortical n e u r o n s (Kawai and W a t a n a b e 16, Sah and McLachlan25). In these cell types, inhibiting Ca 2 + release from intracellular stores shortens the A H P and increasing Ca 2 + release e n h a n c e s the A H P (Kawai and W a t a n a b e 16, Sah and McLachlan25). Similar to the s A H P in neocortical neurons, the Ca-dep e n d e n t K + c o n d u c t a n c e underlying the prolonged A H P in vagal m o t o n e u r o n s is insensitive to apamin and r e d u c e d by NE. In vagal m o t o n e u r o n s , this conductance is decreased by agents which attenuate release from internal Ca 2+ stores (Sah and McLachlan25). A g e - r e l a t e d differences in firing behavior result in a different substrate for modulation. Inputs to developing rat neocortical n e u r o n s a p p e a r to be transduced differently than inputs to m a t u r e cells such that the faithfulness with which prolonged excitatory inputs are converted into action potentials would be diminished in i m m a t u r e cortical neurons relative to adult cells. T h e p r o l o n g e d s A H P may serve to reduce the t e n d e n c y for excitatory inputs to cause sustained depolarizations in i m m a t u r e cells which may not be able to handle large Ca z+ loads. Sustained depolarization and repetitive firing could lead to excessive Ca 2+ influx and p e r h a p s cell death (ChoiS). D u r i n g early postnatal development, the m a m malian neocortex is especially sensitive to changes in functional organization due to e x p e r i e n c e - d e p e n d e n t modifications (Rothblat et al. 24, Wiesel and Hubel3~). Several studies suggest that N E plays a role in ocular d o m i n a n c e plasticity in visual cortex (e.g. Bear and Singer 2, G r e u e l et al. 9, Kasamatsu and Pettigrew ~5) as
well as other forms of neuronal plasticity (for cxample, long-term potentiation in dentate gyrus and CA3 hipp o c a m p a l n e u r o n s : Bliss ct al. 4, H o p k i n s and Johnstonl2). Singer et al. 2v have proposed that a modification threshold involving Ca 2. influx through voltage-gated or N M D A - a c t i v a t e d Ca 2 ~ channels must be reached for such plastic changes to occur in the developing cortex. In this model, the probability of Ca 2+ entry is increased with correlated activity of pre- and post-synaptic neurons (sustained depolarization would o p e n voltage-gated channels or remove Mg 2÷ block of N M D A - a c t i v a t e d channels). Transmitters such as N E could thus affect neuronal plasticity at several levels: (1) increasing or correlating activity; (2) depolarizing the cell; (3) facilitating N M D A responses; (4) facilitating Ca 2+ entry directly t h r o u g h Ca 2+ channels; or (5) decreasing K + conductances, thus indirectly increasing Ca 2+ influx by either route (no. 3 or 4). We have provided evidence for a decrease in a specific Ca-dep e n d e n t K + c o n d u c t a n c e which underlies the s A H P and spike-frequency adaptation in immature neocortical pyramidal neurons. As the s A H P would tend to restrict the duration of Ca 2 + entry and thus Ca-related synaptic plasticity, N E ' s reduction of A H P conductances could indirectly regulate attainment of a modification threshold for developmental synaptic plasticity. Acknowledgements. We thank W. Armstrong and D.J. Surmeier for
valuable comments on an earlier version of this manuscript. This work was supported by National Institute of Neurological Disorders and Stroke Grant NS-27180 (to R.C.F.).
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