Molecular Brain Research, 14 (1992) 207-212 © 1992 Elsevier Science Publishers B.V. All rights reserved. 0169-328X/92/$05.00
207
BRESM 70438
Age-dependent regulation of G A B A A receptors in neocortex Christopher Shaw and Brian Andrew Scarth Departments of Ophthalmology, Physiology and Neuroscience, University of British Columbia, Vancouver, B.C. (Canada) (Accepted 11 February 1992)
Key words: Receptor; GABAA; GABA; Regulation; Cortex; Age dependence
We have shown previously that GABAA receptors labelled with the antagonist [3H]SR95531 can be regulated in a living cortical slice preparation by agonists or changes in electrical activity26. Due to the important role that receptor regulation may play in controlling neural activity, we have now investigated the regulation of GABAA receptors in neocortex at different stages in postnatal life. We found that regulation by agonist stimulation and increases in bioelectric activity is age-dependent in amount and, in the latter case, in direction. Using muscimol as an agonist we observed a GABAA receptor down-regulation of between 60 and 70% at 20-30 days of age', in adults muscimol gave an 11% down-regulation. A combination of veratridine and glutamate gave a peak down-regulation of 39% at 20 days postnatal, but an up-regulation of 58% in adults. These age-dependent effects may signal a role for receptor regulation in cortical neural critical period plasticity. INTRODUCTION Many types of cells respond to increased agonist activation of their receptors by a decrease in the n u m b e r of receptors for that agonist. This regulation, termed 'down-regulation', appears in most cases to result from a decrease in the n u m b e r of receptors 4'9'1°'12A3'19'27'29. Conversely, a receptor 'up-regulation' may occur following treatment with receptor antagonists 6'17'34. Similar types of regulation occur for cells in the brain 2'6'17A9'27' 34. In addition, increases or decreases in the electrical activity of neurons may also lead to a regulation of their various receptors L26'27. Thus the regulation of neurot r a n s m i t t e r / n e u r o m o d u l a t o r (NT/NM) receptors in brain appears to be a p r o m i n e n t receptor characteristic and may play an important role in modulating neural activity. Recently, we have begun to examine the ability of living cells contained within adult rat cortical brain slices to regulate NT/NM receptors. We have demonstrated a decrease in muscarinic acetylcholine receptors ( m A C h R s ) following treatment with carbachol, a m A C h R agonist, and following treatment with veratridine, a cellular depolarizing agent 27. In the same manner, we have investigated the effects of v-aminobutyric acid ( G A B A ) and muscimol, a G A B A A receptor agonist, and veratridine on G A B A A receptors. In the first case, G A B A and muscimol led to decreases in G A B A A receptor n u m b e r 26. In contrast to the decrease for
m A C h R s seen following veratridine, G A B A A receptors were greatly increased in n u m b e r by a combined veratridine plus glutamate treatment z6. Preliminary data for a n u m b e r of other cortical receptor populations suggests that these may be regulated as well by appropriate agonist stimulation or changes in cell electrical activity (e.g., nicotinic A C h and adenosine receptors; Shaw et al., unpublished results). Given the tendency of cortical cells to show receptor regulation, we were curious to see if such regulation was a consistent feature at different postnatal ages, especially within one of the physiologically defined critical periods for n e u r o n a l plasticity 11'15'22. The present results show that not only is the G A B A A receptor in neocortex regulated, but that both the a m o u n t and even direction of the regulation are age-dependent. MATERIALS AND METHODS All animals included in the present study were colony bred rats (Sprague-Dawley) or cats maintained in the same light-dark cycle (12:12 h) and sacrificed at approximately the same time of day (rats, 09.00-10.00 h; cats, 11.00-12.00 h). Rat ages varied between 5 days and more than i00 days postnatal. Most rats below the age of puberty (approx. 35 days) were males and all rats after this age were males. In the cat experiments, only 23 day old was a female. All animals were initially deeply anesthetized with halothane and then decapitated. In all of these experiments on both species, the brains were removed in under 1 min and placed in a modified Dulbecco's medium (for complete details see ref. 32). This medium, referred to as Dul +, was found to retain cell viability for a number of hours and was used in all subsequent treatment. For rats, blocks of neocortex containing various cortical f i e l d s 16'26"32 w e r e dissected
Correspondence: C. Shaw, Department of Ophthalmology, c/o Department of Anatomy, 2177 Wesbrook Mall, Vancouver, B.C., V6T 1Z3 Canada.
208 out and sectioned at 400 ~m thickness using a device described elsewhere 3. In cats, only visual cortex areas 17-19 were taken 3~. These latter blocks were dissected into a medial bank (area 17) block and, where possible, a 17/18 border to area 19 block. In both types of block the pia was removed. Slices obtained in both cases were transferred to tissue culture wells containing 0.5 ml Dul +. In general, the conditions for each row of slices were repeated 2-4 times per experiment. Full details of the characterization of the GABA A receptor in this preparation using [3H]SR95531 have been reported previously26. In brief, slices were exposed to a combination of veratridine and glutamate (v+g) or muscimol for 2 h at 37°C, then rinsed 2 × for 30 min before incubation with [3H]SR95531 for 90 min at 4°C. (Note in regard to these 2 x 30 min rinses that we have shown previously that rinses of this duration are sufficient to remove all the remaining muscimol or veratridine at the various ages (data not shown for animals younger than adult; for adults see ref. 26). In the former case it is obvious that regulation experiments must ensure that no agonist remains in the well prior to the incubation with the radioligand. In the second case, a competition of veratridine for the GABA A receptor labelled with [3H]SR95531 does occur at the different ages. This result is not completely unexpected since we have reported elsewhere that a related alkaloid can displace opioid and muscarinic receptor binding33.) For regulation experiments low concentrations of ligand (5 mM) were usually chosen for reasons of economy since saturation binding experiments showed that agonist- or v+g-induced changes reflected Bma× rather than Kd alterations. The slices were rinsed for 2 x 5 rain in Dul + at 4°C. Following the final rinses in 4°C Dul +. the slices were picked up with Whatman GF/B glass fiber filter paper and placed in vials containing NEN Formula 963 scintillation fluid. Racks containing the samples were kept in the dark for a minimum of 12 h and then counted in a scintillation counter (Beckman LS 6000IC with an efficiency of approximately 55%). For each individual experiment, the effects of adding the various drugs were calculated as the percent difference from radioligand bound in control slices 27. Data were analyzed for significance using a one sample t-test (one-tailed). Receptor binding values following the various manipulations (Fig. I) are expressed as percent difference from control GABA A receptor number _+ S.E.M. for at least 6 separate experiments.
T h e greatest d o w n - r e g u l a t i o n was - 3 9 % at 20 days postnatal. A f t e r 35 days postnatal, v + g t r e a t m e n t always resuited in an u p - r e g u l a t i o n of G A B A A r e c e p t o r s with a p e a k of + 9 8 % at 42 days and a s u b s e q u e n t decline to + 5 8 % in adults. F r o m 26 days postnatal until approxim a t e l y 35 days postnatal, the direction and m a g n i t u d e of r e g u l a t i o n w e r e quite variable a l t h o u g h the net effect t e n d e d to reflect a relatively s m o o t h l y changing direction of regulation. Significant differences f r o m control w e r e o b s e r v e d (at least P < 0.05) for all points after 5 days postnatal age. Muscimol
(Fig.
2B)
led to a significant
the a m o u n t of such regulation was a g e - d e p e n d e n t , with a n e g a t i v e t r o u g h of - 6 4 % to - 6 8 % at a p p r o x i m a t e l y 2 0 - 3 0 days postnatal, declining to - 1 1 % in a d u l t h o o d . T h e change in the a m o u n t a n d / o r direction of regulation at the different ages following v + g or m u s c i m o l t r e a t m e n t was a c o n s e q u e n c e of a c h a n g e in G A B A A r e c e p t o r n u m b e r . S a t u r a t i o n binding e x p e r i m e n t s at 20 days postnatal and in adult cortical slices (Fig. 2 A , B , insets) r e v e a l e d a d e c r e a s e in B ..... for v + g and agonist at 20 days and an increase in Bm~ ~ for v + g in adults with no c h a n g e in the affinity of the r e c e p t o r at these ages. Preliminary
results
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Fig. 1 shows the effects of G A B A
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d o w n - r e g u l a t i o n at all ages (at least P < 0.05), a l t h o u g h
agonists or of in-
creases in b i o e l e c t r i c activity p r o d u c e d by the a d d i t i o n of v e r a t r i d i n e plus g l u t a m a t e ( v + g ) on G A B A A receptors in adult rat n e o c o r t e x . A s we h a v e r e p o r t e d previously 26, such t r e a t m e n t s led to statistically significant
20
o ol
0
¢1 .¢
O
-20
-40
d o w n - or u p - r e g u l a t i o n of the G A B A A r e c e p t o r , respectively. O f the agonists, m u s c i m o l was a m o r e effective agent for inducing d o w n - r e g u l a t i o n of the G A B A A rec e p t o r in the cortical slice p r e p a r a t i o n than G A B A . Baciofen, which is selective for G A B A B r e c e p t o r s , was w i t h o u t significant effect. O n l y m u s c i m o l and v + g treatm e n t g a v e statistically significant effects (t-test) o f - 1 1 % ( P < 0.05) and + 5 8 % ( P < 0.001), respectively. In Fig. 2 we s h o w the effects of v + g or m u s c i m o l t r e a t m e n t on slices f r o m rats of d i f f e r e n t postnatal ages. T h e effects o b s e r v e d with v + g t r e a t m e n t (Fig. 2 A ) s h o w e d a r e v e r s a l in the d i r e c t i o n of r e g u l a t i o n with age: A t ages b e t w e e n 10 and 26 days postnatal, v + g treatm e n t i n d u c e d a d o w n - r e g u l a t i o n of G A B A A r e c e p t o r s .
X
nn
e--
Fig. 1. Regulation of the GABA A receptor in adult rat neocortex labelled with [3H]SR95531. Various substances were added to cortical slices in separate rows of wells for up to 2 h at 37°C and compared to control slices. The substances included: GABA, and the GABA A and GABA B agonists muscimol and baclofen, respectively (final concentrations, 10-5 M), agents designed to increase cell depolarization and neural activity (veratridine plus glutamate, at 10-5 M). Slices were rinsed 2 x 30 min at 4°C, then incubated at 4°C with 5 nM [3H]SR95531 for 90 min to 2 h. Typically for each row of 5 slices representing each condition, 3 slices were used to determine total binding. Non-specific binding was determined in 2 other slices by co-incubating with 10-4 M GABA. A one-tailed t-test was utilized in data analysis: *P < 0.05, **P < 0.001.
209
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Fig. 2. Age-dependent regulation of the GABA A receptor by veratridine+glutamate (v+g) and muscimol. The effects of v+g (A) or muscimol (B) were investigated in cortical slices from rats of different postnatal ages ranging from 5 days to adult (>70-100 days postnatal age). In both A and B, the abscissa shows postnatal age (in days) while the ordinate shows the percentage difference from control slices for [aH]SR95531 binding. In all of these experiments, the [3H]SR95531 concentration was 5 nM. In several instances, saturation binding analysis was performed as described previously26, but separately for control (open circles) and v+g treated slices (squares). The two inset panels to A and single inset in B show Eadie-Hofstee plots of individual saturation binding assays in rat neocortex using [3H]SR95531 following v+g or muscimol treatment, respectively, for cortical slices from adult and 20 day old rats. In such cases, additional sections were assayed for protein TM and the resulting binding data expressed conventionally as fmol/mg protein. All Bma× and K d determinations were made as described elsewhere35. In A, the uppermost inset is for the adult animals; the inset below is for animals at 20 days of age. In B, the inset is for the 20 day old animals alone since the effect of muscimol (11) vs control (O) at this age is relatively large. In the 20 day old animals it is apparent that both v+g and muscimol treatments led to a loss of GABA A receptor numbers with no change in receptor affinity (Bmax change of -40% and -42%, respectively). Conversely, in adult animals GABA A receptors were up-regulated by v+g treatment (Bm, ~ change of +35%) with no change in affinity. S.E.M. values in the graphs of A and B are for at least 5 experiments/age.
210 The E a d i e - H o f s t e e plots of the saturation binding data from rat cortical slices are linear (Fig. 2), showing only a single population of G A B A A receptors at these ages. Similarly, only single G A B A A receptor populations were present at the other ages examined (data not shown for all ages). Data representing B .... and K a values for a n u m b e r of different postnatal ages are plotted in Fig. 3A and B, respectively, Peak G A B A A receptor number was found at about 30 days postnatal with a corresponding peak in K d. These results are consistent with developmental changes we have observed previously in the visual cortex of other species 2>25.
A 4000
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Previous studies have shown changes in the amount of receptor regulation as a function of age 8't4'2~'~°, although we believe that the present results are the first to report changes in the direction of regulation. That the G A B A A receptor shows such effects may be particularly notable in light of the inhibitory role that G A B A appears to play in cortical information processing 28 and for the role it may play in plastic modifications of cortical function 2.24.
Age (days)
B 80
60 A ¢v
t
40
"O v
20
20
40
60
80
A g e (days)
Fig. 3. Development of GABA A receptor characteristics in rat neocortex. [3H]SR95531 saturation binding experiments were performed on rat cortical slices at different postnatal ages. The postnatal development of receptor number (Bma×) (A) and affinity (Kd) (B) as a function of postnatal age are shown for ages from 10 to 70 days. Note the corresponding peak in receptor number with the lowest affinity at about 30 days postnatal. S.E.M. values are for single saturation binding assays at each age (representing 2-5 animals/experiment) except for the determination at 39 days which represents the combinded data of two experiments.
bank, area 17) were qualitatively similar to those obtained for rat neocortex: at 55 days postnatal v + g increased [3H]SR95531 binding by 10% while muscimol decreased binding by 31%; at 30 days both v + g and muscimol decreased binding by 20% and 48%, respectively; at 23 days v + g and muscimol decreased binding by 24% and 65%, respectively; at 16 days v + g and muscimol decreased binding by 8% and 71%, respectively.
The qualitative similarity in the two different species r e p o r t e d here, utilizing different regions of neocortex, may suggest that some aspects of the regulation of G A B A a receptors induced by agonist or changes in bioelectric activity are general features of neocortex in postnatal development. For rat neocortex, the significance of the apparent peak in G A B A A receptor number corresponding to the period in postnatal life in which such receptors shift their direction of regulation in response to changes in electrical stimulation is not known. We have noted elsewhere, however, a modification of muscarinic ACh receptor characteristics near this age for orchiectom i z e d / o v a r i e c t o m i z e d rats 2°. This observation may suggest that sex h o r m o m e s play a role in some aspects of receptor development. One implication of the present study is that changes in input activity might be dealt with differentially by mechanisms of receptor regulation. Thus, for example, increased input activity, such as might be associated with the induction of long-term potentiation (LTP) (which has also been shown to be age-dependent in rat 22 and cat visual cortex~S), might be expected to lead to a decrease in G A B A A receptor number at the affected synapses at certain ages. (N.B., LTP is usually induced in large numbers of neurons simultaneously, similar to our mass manipulation by bath-applied muscimol or v + g of G A B A A receptor regulation). If such a decrease in inhibitory receptors were coupled to an increase in excitatory receptor number, it is possible to imagine how synaptic
211 strengthening might occur. Conversely, increased input activity in similar circuits in adult cortex would a p p e a r to give an increase in G A B A A r e c e p t o r n u m b e r and perhaps a concomitant increase in inhibition at that synapse. If such activity also caused a down-regulation of excitatory receptors, as it does for m o d u l a t o r y receptors 27' then the net consequence might be a ' d a m p i n g ' of synaptic activity acting to prevent long-term changes in synaptic activity. A s a working hypothesis we propose that various forms of cortical plasticity result from the c o m b i n e d weight of inhibitory and excitatory synaptic activity, differentially regulated by their various r e c e p t o r populations during early stages of postnatal life. Thus neuronal plasticity will occur following changes in neuronal activity i f there is a net decrease in inhibitory r e c e p t o r n u m b e r and, perhaps, an increase in excitatory r e c e p t o r number. The p e r i o d in which such changes are the most p r o m i n e n t would then constitute the p e a k of the 'critical period'. It is worth noting in this regard that the p e a k for inducing LTP in rat visual cortex appears to be near 15 days postnatal 22, close to our o b s e r v e d m a x i m u m in G A B A A receptor down-regulation. In addition, the peaks for both LTP induction in cat visual cortex (21-34 days postnatal 15) as well as for m o n o c u l a r deprivation plasticity (near 30 days) 11 occur m o r e or less coincidentally with the m a x i m u m G A B A A r e c e p t o r down-regulation to altered neural activity r e p o r t e d here in our cat data. In regard to m o n o c u l a r deprivation however, it is clear that such effects are quite complex and may not be easily c o m p a r e d to cortical LTP since they involve several stages, one of which ,may be the loss of a fraction of the terminals representing the closed eye (see ref. 7). In this m o d e l we further speculate that as the direction of regulation begins to shift for the various receptors with age, the ' e n v e l o p e ' of regulation closes and the critical p e r i o d comes to an end. We note that the only way that cortical LTP can be routinely observed in adult animals involves the experimental reduction in functional G A B A A receptors with bicuculline 2.
REFERENCES 1 Abdul Ghani, A.S., Boyer, M.M., Coutinho-Neno, J., Bradford, H.F., Benie, C.P., Hulme, E.C. and Birdsall, N.J.M., Effect of tityus toxin and sensory stimulation on muscarinic cholinergic receptors in vivo, Biochem. Pharmacol., 30 (1981) 27132714. 2 Artola, A. and Singer, W., Long-term potentiation and NMDA receptors in rat visual cortex, Nature, 330 (1987) 649-652. 3 Bennett, G.W., Sharp, T., Marsden, C.A. and Parker, T.L., A manually-operated brain tissue slicer suitable for neurotransmitter release studies, J. Neurosci. Methods, 7 (1983) 107-115. 4 Bloch, R.J., Loss of acetylcholine receptor clusters induced by treatment of cultured rat myotubes with carbacbol, J. Neurosci., 6 (1986) 691-700.
Such a m o d e l requires much new information before it can be accepted. We do not know that excitatory receptors show reciprocal regulation to the G A B A A receptors in cortex and it is likely naive to assume that they will do so in all cases. Studies in progress are directed at this issue. F u r t h e r m o r e , the underlying mechanisms leading to G A B A A r e c e p t o r regulation at any age, let alone the a g e - d e p e n d a n t regulation we have rep o r t e d here, are not completely understood. Some aspects of such regulation a p p e a r to be related to the activity of r e c e p t o r kinases and phosphatases, leading to changes in the n u m b e r of functional G A B A A receptors via d e p h o s p h o r y l a t i o n reactions 5. If such were the case, it would be a simple and attractive mechanism for neuronal regulation if similar kinase/phosphatase reactions had reciprocal effects and/or different concentration dependencies on excitatory receptors such as the A M P A and N M D A subtypes. F u r t h e r m o r e , differential age-dep e n d e n t regulation of inhibitory and excitatory receptors might be m e d i a t e d by a g e - d e p e n d e n t kinases and phosphatases. While much of the foregoing is highly speculative, we believe that the present quantitative data suggest novel ways in which cortical r e c e p t o r populations may be altered and, in consequence of which, how alterations in synaptic function m a y occur. Future studies, perhaps using electrophysiological techniques c o m b i n e d with receptor assays, may open new vistas to one of the most pressing problems in the neurosciences, i.e., the restoration of plasticity in the brain after the normal term of the critical period.
Acknowledgements. The present studies were supported grants from the Canadian Medical Research Council, the British Columbia Health Care Research Foundation and the Adamson Foundation to C.S.C.S. is a BC Health Care Research Scholar. B.A.S. is the recipient of an NSERC Summer Fellowship. We thank B.A. Pasqualotto and R.A. Lanius for assistance and Drs. M. Wilkinson, J. Simmons and E Van Huizen for helpful comments on the manuscript.
5 Chen, Q.X., Stelzer, A., Kay, A. and Wong, R., GABA A receptor function is regulated by phosphorylation in acutely dissociated guinea-pig hippocampal neurones, J. Physiol., 420 (1990) 207-221. 6 EI-Fakahany, E.E. and Lee, J.H., Agonist-induced muscarinic acetylcholine receptor down-regulation in intact rat brain cells, Eur. J. Pharmacol., 132 (1986) 21-30. 7 Gordon, B., Allen, E.E. and Trombley, P.Q., The role of norepinephrine in plasticity of visual cortex, Prog. Neurobiol., 30 (1988) 171-191. 8 Handelmann, G. and Quirion, R., Neonatal exposure to morphine increases ~ opiate binding in the adult forebrain, Eur. J. Pharmacol., 94 (1983) 357-358. 9 Hollenberg, M.D., Biochemical mechanisms of receptor regulation, Trends Pharmacol. Sci., 6 (1985) 299-302.
212 10 Hollenberg, M.D., Control of receptor function by homologous and heterologous ligands. In G. Poste and S.T. Crooke (Eds.), Mechanisms of Receptor Regulation, Plenum, New York, 1985, pp. 295-322. 11 Hubel. D.H. and Wiesel, T.N., The period of susceptibility to the physiological effects of unilateral eye closure in kittens, J. Physiol., 206 (1970) 419-436. i2 Hunter, D.D. and Nathanson, N.M., Biochemical and physical analysis of newly synthesized muscarinic acetylcholine receptors on cultured embryonic chicken cardiac cells, J. Neurosci., 6 (1986) 3739-3748. 13 Jarett, L. and Smith R.M., Receptor regulation, problems and perspectives. In G. Poste and S.T. Crooke (Eds.), Mechanisms of Receptor Regulation, Plenum, New York, 1985, pp. 1-11. 14 Kochman, K., Joseph, J.A., Blackman, M.R. and Stagg, C.A.. Impaired down-regulation of pituitary dopamine receptors by estradiol in aged rats, Proc. Soc. Exp. Biol. Med., 192 (1989) 23-26. 15 Komatsu, Y., Fujii, K., Maeda, J., Sakaguchi, H. and Toyama, K., Long-term potentiation of synaptic transmission in kitten visual cortex, J. Neurophysiol., 59 (1988) 124-141. 16 Krieg, W.J.S., Connections of the cerebral cortex. I. The albino rat. A topography of the cortical areas, J. Comp. Neurol., 84 (1946) 221-275. 17 Liles, W.C. and Nathanson, N.M., Regulation of acetylcholine receptor number in cultured neuronal cells by chronic membrane depolarization, J. Neurosci., 7 (1987) 2556-2563. 18 Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J., Protein measurement with the Folin phenol reagent, J. Biol. Chem., 193 (1951) 265-275. 19 Maloteaux, J., Octave, J., Gossuin, A., Laterre, C. and Trouet, A., GABA induces down-regulation of the benzodiazepineGABA receptor complex in the rat cultured neurons, Eur. J. Pharmacol., 144 (1989) 173-183. 20 March, D., Van Huizen, F. and Shaw, C., Effects of testosterone and estrogen on muscarinic receptor binding in rat cerebral cortex slices, Soc. Neurosci. Abstr., 14 (1988) 270. 21 Meaney, M.J., Sapolsky, R.M. and McEwen, B.S., The development of the glucocorticoid receptor system in the rat limbic brain. II. An autoradiographic study, Dev. Brain Res., 18 (1985) 165-168. 22 Perkins, A.T. and Teyler, T.J.. A critical period for long-term potentiation in the developing rat visual cortex, Brain Res.. 439 (1988) 222-229. 23 Shaw, C., Cameron, L., March, D., Cynader, M., Zielinski, B,
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and Hendrickson, A., Pre- and postnatal development of GABA receptors in Macaca, monkey visual cortex, J, Neurosci., ll (1991) 3943-3959. Shaw, C. and Cynader, M.. Unilateral eyelid suture increases G A B A A receptors in cat visual cortex, Dev. Brain Res., 40 (1988) 148-153. Shaw, C., Needler. M.C. and Cynader, M., Ontogenesis of muscimol binding sites in cat visual cortex. Brain Res. Bull., 13 (1984) 331-334. Shaw, C. and Scarth, B.A., Characterization and differential regulation of GABA A and benzodiazepine receptors in rat neocortex, Mol. Brain Res., 11 (1991) 273-282. Shaw, C., Van Huizen, F.. Cynader, M. and Wilkinson, M., A role for potassium channels in the regulation of cortical muscarinic acetylcholine receptors in an in vitro slice preparation, Mol. Brain Res., 5 (19891 71-83. Sillito, A.M., Inhibitory circuits and orientation selectivity in the visual cortex. In D. Rose and V.G. Dobson (Eds.), Models of the Visual Cortex, Wiley, London, 1985, pp. 396-407. Stollberg, J. and Berg, D.K., Neuronal acetylcholine receptors: fate of surface and internal pools in cell culture, J. Neurosci., 7 (1987) 1809-1815. Turner, N.. Houck, W.T. and Roberts, J., Effect of age on upregulation of the cardiac adrenergic fl-receptors, J. Gerontol.. 45 (1990) B48-51, Tusa, R.J., Palmer, L.A. and Rosenquist, A.C., Multiple cortical visual areas: visual field topography in the cat. In C.N. Woolsey (Ed.), Cortical Senso O, Organization, Vol. 2, Humana, Clifton, NJ, 1981, pp. 1-31, Van Huizen, F., Shaw, C.. Wilkinson, M. and Cynader, M., Characterization of muscarinic acetylcholine receptors in rat cerebral cortex slices with concomitant morphological and physiological assessment of tissue viability, Mol. Brain Res., 5 (1989) 59-69. Van Huizen, E, Wilkinson, M., Cynader, M. and Shaw, C., Sodium channel toxins veratrine and veratridine modify opioid and muscarinic but not fl-adrenergic binding sites in brain slices, Brain Res. Bull., 40 (1988) 129-132. Yamada, S., Isogai, M., Okudaira, H. and Ash, E.H., Regional adaptation of muscarinic receptors and choline uptake in brain following repeated administration of diisopropyl-fluorophosphate and atropine, Brain Res.. 268 (1983) 315-32fl. Zivin, J.A. and Waud, D.R., How to analyse binding, enzyme and uptake data: the simplest case, a single phase, Life Sci.. 30 (1982) 1407-1422.
207
BRESM 70438
Age-dependent regulation of G A B A A receptors in neocortex Christopher Shaw and Brian Andrew Scarth Departments of Ophthalmology, Physiology and Neuroscience, University of British Columbia, Vancouver, B.C. (Canada) (Accepted 11 February 1992)
Key words: Receptor; GABAA; GABA; Regulation; Cortex; Age dependence
We have shown previously that GABAA receptors labelled with the antagonist [3H]SR95531 can be regulated in a living cortical slice preparation by agonists or changes in electrical activity26. Due to the important role that receptor regulation may play in controlling neural activity, we have now investigated the regulation of GABAA receptors in neocortex at different stages in postnatal life. We found that regulation by agonist stimulation and increases in bioelectric activity is age-dependent in amount and, in the latter case, in direction. Using muscimol as an agonist we observed a GABAA receptor down-regulation of between 60 and 70% at 20-30 days of age', in adults muscimol gave an 11% down-regulation. A combination of veratridine and glutamate gave a peak down-regulation of 39% at 20 days postnatal, but an up-regulation of 58% in adults. These age-dependent effects may signal a role for receptor regulation in cortical neural critical period plasticity. INTRODUCTION Many types of cells respond to increased agonist activation of their receptors by a decrease in the n u m b e r of receptors for that agonist. This regulation, termed 'down-regulation', appears in most cases to result from a decrease in the n u m b e r of receptors 4'9'1°'12A3'19'27'29. Conversely, a receptor 'up-regulation' may occur following treatment with receptor antagonists 6'17'34. Similar types of regulation occur for cells in the brain 2'6'17A9'27' 34. In addition, increases or decreases in the electrical activity of neurons may also lead to a regulation of their various receptors L26'27. Thus the regulation of neurot r a n s m i t t e r / n e u r o m o d u l a t o r (NT/NM) receptors in brain appears to be a p r o m i n e n t receptor characteristic and may play an important role in modulating neural activity. Recently, we have begun to examine the ability of living cells contained within adult rat cortical brain slices to regulate NT/NM receptors. We have demonstrated a decrease in muscarinic acetylcholine receptors ( m A C h R s ) following treatment with carbachol, a m A C h R agonist, and following treatment with veratridine, a cellular depolarizing agent 27. In the same manner, we have investigated the effects of v-aminobutyric acid ( G A B A ) and muscimol, a G A B A A receptor agonist, and veratridine on G A B A A receptors. In the first case, G A B A and muscimol led to decreases in G A B A A receptor n u m b e r 26. In contrast to the decrease for
m A C h R s seen following veratridine, G A B A A receptors were greatly increased in n u m b e r by a combined veratridine plus glutamate treatment z6. Preliminary data for a n u m b e r of other cortical receptor populations suggests that these may be regulated as well by appropriate agonist stimulation or changes in cell electrical activity (e.g., nicotinic A C h and adenosine receptors; Shaw et al., unpublished results). Given the tendency of cortical cells to show receptor regulation, we were curious to see if such regulation was a consistent feature at different postnatal ages, especially within one of the physiologically defined critical periods for n e u r o n a l plasticity 11'15'22. The present results show that not only is the G A B A A receptor in neocortex regulated, but that both the a m o u n t and even direction of the regulation are age-dependent. MATERIALS AND METHODS All animals included in the present study were colony bred rats (Sprague-Dawley) or cats maintained in the same light-dark cycle (12:12 h) and sacrificed at approximately the same time of day (rats, 09.00-10.00 h; cats, 11.00-12.00 h). Rat ages varied between 5 days and more than i00 days postnatal. Most rats below the age of puberty (approx. 35 days) were males and all rats after this age were males. In the cat experiments, only 23 day old was a female. All animals were initially deeply anesthetized with halothane and then decapitated. In all of these experiments on both species, the brains were removed in under 1 min and placed in a modified Dulbecco's medium (for complete details see ref. 32). This medium, referred to as Dul +, was found to retain cell viability for a number of hours and was used in all subsequent treatment. For rats, blocks of neocortex containing various cortical f i e l d s 16'26"32 w e r e dissected
Correspondence: C. Shaw, Department of Ophthalmology, c/o Department of Anatomy, 2177 Wesbrook Mall, Vancouver, B.C., V6T 1Z3 Canada.
208 out and sectioned at 400 ~m thickness using a device described elsewhere 3. In cats, only visual cortex areas 17-19 were taken 3~. These latter blocks were dissected into a medial bank (area 17) block and, where possible, a 17/18 border to area 19 block. In both types of block the pia was removed. Slices obtained in both cases were transferred to tissue culture wells containing 0.5 ml Dul +. In general, the conditions for each row of slices were repeated 2-4 times per experiment. Full details of the characterization of the GABA A receptor in this preparation using [3H]SR95531 have been reported previously26. In brief, slices were exposed to a combination of veratridine and glutamate (v+g) or muscimol for 2 h at 37°C, then rinsed 2 × for 30 min before incubation with [3H]SR95531 for 90 min at 4°C. (Note in regard to these 2 x 30 min rinses that we have shown previously that rinses of this duration are sufficient to remove all the remaining muscimol or veratridine at the various ages (data not shown for animals younger than adult; for adults see ref. 26). In the former case it is obvious that regulation experiments must ensure that no agonist remains in the well prior to the incubation with the radioligand. In the second case, a competition of veratridine for the GABA A receptor labelled with [3H]SR95531 does occur at the different ages. This result is not completely unexpected since we have reported elsewhere that a related alkaloid can displace opioid and muscarinic receptor binding33.) For regulation experiments low concentrations of ligand (5 mM) were usually chosen for reasons of economy since saturation binding experiments showed that agonist- or v+g-induced changes reflected Bma× rather than Kd alterations. The slices were rinsed for 2 x 5 rain in Dul + at 4°C. Following the final rinses in 4°C Dul +. the slices were picked up with Whatman GF/B glass fiber filter paper and placed in vials containing NEN Formula 963 scintillation fluid. Racks containing the samples were kept in the dark for a minimum of 12 h and then counted in a scintillation counter (Beckman LS 6000IC with an efficiency of approximately 55%). For each individual experiment, the effects of adding the various drugs were calculated as the percent difference from radioligand bound in control slices 27. Data were analyzed for significance using a one sample t-test (one-tailed). Receptor binding values following the various manipulations (Fig. I) are expressed as percent difference from control GABA A receptor number _+ S.E.M. for at least 6 separate experiments.
T h e greatest d o w n - r e g u l a t i o n was - 3 9 % at 20 days postnatal. A f t e r 35 days postnatal, v + g t r e a t m e n t always resuited in an u p - r e g u l a t i o n of G A B A A r e c e p t o r s with a p e a k of + 9 8 % at 42 days and a s u b s e q u e n t decline to + 5 8 % in adults. F r o m 26 days postnatal until approxim a t e l y 35 days postnatal, the direction and m a g n i t u d e of r e g u l a t i o n w e r e quite variable a l t h o u g h the net effect t e n d e d to reflect a relatively s m o o t h l y changing direction of regulation. Significant differences f r o m control w e r e o b s e r v e d (at least P < 0.05) for all points after 5 days postnatal age. Muscimol
(Fig.
2B)
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the a m o u n t of such regulation was a g e - d e p e n d e n t , with a n e g a t i v e t r o u g h of - 6 4 % to - 6 8 % at a p p r o x i m a t e l y 2 0 - 3 0 days postnatal, declining to - 1 1 % in a d u l t h o o d . T h e change in the a m o u n t a n d / o r direction of regulation at the different ages following v + g or m u s c i m o l t r e a t m e n t was a c o n s e q u e n c e of a c h a n g e in G A B A A r e c e p t o r n u m b e r . S a t u r a t i o n binding e x p e r i m e n t s at 20 days postnatal and in adult cortical slices (Fig. 2 A , B , insets) r e v e a l e d a d e c r e a s e in B ..... for v + g and agonist at 20 days and an increase in Bm~ ~ for v + g in adults with no c h a n g e in the affinity of the r e c e p t o r at these ages. Preliminary
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creases in b i o e l e c t r i c activity p r o d u c e d by the a d d i t i o n of v e r a t r i d i n e plus g l u t a m a t e ( v + g ) on G A B A A receptors in adult rat n e o c o r t e x . A s we h a v e r e p o r t e d previously 26, such t r e a t m e n t s led to statistically significant
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d o w n - or u p - r e g u l a t i o n of the G A B A A r e c e p t o r , respectively. O f the agonists, m u s c i m o l was a m o r e effective agent for inducing d o w n - r e g u l a t i o n of the G A B A A rec e p t o r in the cortical slice p r e p a r a t i o n than G A B A . Baciofen, which is selective for G A B A B r e c e p t o r s , was w i t h o u t significant effect. O n l y m u s c i m o l and v + g treatm e n t g a v e statistically significant effects (t-test) o f - 1 1 % ( P < 0.05) and + 5 8 % ( P < 0.001), respectively. In Fig. 2 we s h o w the effects of v + g or m u s c i m o l t r e a t m e n t on slices f r o m rats of d i f f e r e n t postnatal ages. T h e effects o b s e r v e d with v + g t r e a t m e n t (Fig. 2 A ) s h o w e d a r e v e r s a l in the d i r e c t i o n of r e g u l a t i o n with age: A t ages b e t w e e n 10 and 26 days postnatal, v + g treatm e n t i n d u c e d a d o w n - r e g u l a t i o n of G A B A A r e c e p t o r s .
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Fig. 1. Regulation of the GABA A receptor in adult rat neocortex labelled with [3H]SR95531. Various substances were added to cortical slices in separate rows of wells for up to 2 h at 37°C and compared to control slices. The substances included: GABA, and the GABA A and GABA B agonists muscimol and baclofen, respectively (final concentrations, 10-5 M), agents designed to increase cell depolarization and neural activity (veratridine plus glutamate, at 10-5 M). Slices were rinsed 2 x 30 min at 4°C, then incubated at 4°C with 5 nM [3H]SR95531 for 90 min to 2 h. Typically for each row of 5 slices representing each condition, 3 slices were used to determine total binding. Non-specific binding was determined in 2 other slices by co-incubating with 10-4 M GABA. A one-tailed t-test was utilized in data analysis: *P < 0.05, **P < 0.001.
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Fig. 2. Age-dependent regulation of the GABA A receptor by veratridine+glutamate (v+g) and muscimol. The effects of v+g (A) or muscimol (B) were investigated in cortical slices from rats of different postnatal ages ranging from 5 days to adult (>70-100 days postnatal age). In both A and B, the abscissa shows postnatal age (in days) while the ordinate shows the percentage difference from control slices for [aH]SR95531 binding. In all of these experiments, the [3H]SR95531 concentration was 5 nM. In several instances, saturation binding analysis was performed as described previously26, but separately for control (open circles) and v+g treated slices (squares). The two inset panels to A and single inset in B show Eadie-Hofstee plots of individual saturation binding assays in rat neocortex using [3H]SR95531 following v+g or muscimol treatment, respectively, for cortical slices from adult and 20 day old rats. In such cases, additional sections were assayed for protein TM and the resulting binding data expressed conventionally as fmol/mg protein. All Bma× and K d determinations were made as described elsewhere35. In A, the uppermost inset is for the adult animals; the inset below is for animals at 20 days of age. In B, the inset is for the 20 day old animals alone since the effect of muscimol (11) vs control (O) at this age is relatively large. In the 20 day old animals it is apparent that both v+g and muscimol treatments led to a loss of GABA A receptor numbers with no change in receptor affinity (Bmax change of -40% and -42%, respectively). Conversely, in adult animals GABA A receptors were up-regulated by v+g treatment (Bm, ~ change of +35%) with no change in affinity. S.E.M. values in the graphs of A and B are for at least 5 experiments/age.
210 The E a d i e - H o f s t e e plots of the saturation binding data from rat cortical slices are linear (Fig. 2), showing only a single population of G A B A A receptors at these ages. Similarly, only single G A B A A receptor populations were present at the other ages examined (data not shown for all ages). Data representing B .... and K a values for a n u m b e r of different postnatal ages are plotted in Fig. 3A and B, respectively, Peak G A B A A receptor number was found at about 30 days postnatal with a corresponding peak in K d. These results are consistent with developmental changes we have observed previously in the visual cortex of other species 2>25.
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Previous studies have shown changes in the amount of receptor regulation as a function of age 8't4'2~'~°, although we believe that the present results are the first to report changes in the direction of regulation. That the G A B A A receptor shows such effects may be particularly notable in light of the inhibitory role that G A B A appears to play in cortical information processing 28 and for the role it may play in plastic modifications of cortical function 2.24.
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Fig. 3. Development of GABA A receptor characteristics in rat neocortex. [3H]SR95531 saturation binding experiments were performed on rat cortical slices at different postnatal ages. The postnatal development of receptor number (Bma×) (A) and affinity (Kd) (B) as a function of postnatal age are shown for ages from 10 to 70 days. Note the corresponding peak in receptor number with the lowest affinity at about 30 days postnatal. S.E.M. values are for single saturation binding assays at each age (representing 2-5 animals/experiment) except for the determination at 39 days which represents the combinded data of two experiments.
bank, area 17) were qualitatively similar to those obtained for rat neocortex: at 55 days postnatal v + g increased [3H]SR95531 binding by 10% while muscimol decreased binding by 31%; at 30 days both v + g and muscimol decreased binding by 20% and 48%, respectively; at 23 days v + g and muscimol decreased binding by 24% and 65%, respectively; at 16 days v + g and muscimol decreased binding by 8% and 71%, respectively.
The qualitative similarity in the two different species r e p o r t e d here, utilizing different regions of neocortex, may suggest that some aspects of the regulation of G A B A a receptors induced by agonist or changes in bioelectric activity are general features of neocortex in postnatal development. For rat neocortex, the significance of the apparent peak in G A B A A receptor number corresponding to the period in postnatal life in which such receptors shift their direction of regulation in response to changes in electrical stimulation is not known. We have noted elsewhere, however, a modification of muscarinic ACh receptor characteristics near this age for orchiectom i z e d / o v a r i e c t o m i z e d rats 2°. This observation may suggest that sex h o r m o m e s play a role in some aspects of receptor development. One implication of the present study is that changes in input activity might be dealt with differentially by mechanisms of receptor regulation. Thus, for example, increased input activity, such as might be associated with the induction of long-term potentiation (LTP) (which has also been shown to be age-dependent in rat 22 and cat visual cortex~S), might be expected to lead to a decrease in G A B A A receptor number at the affected synapses at certain ages. (N.B., LTP is usually induced in large numbers of neurons simultaneously, similar to our mass manipulation by bath-applied muscimol or v + g of G A B A A receptor regulation). If such a decrease in inhibitory receptors were coupled to an increase in excitatory receptor number, it is possible to imagine how synaptic
211 strengthening might occur. Conversely, increased input activity in similar circuits in adult cortex would a p p e a r to give an increase in G A B A A r e c e p t o r n u m b e r and perhaps a concomitant increase in inhibition at that synapse. If such activity also caused a down-regulation of excitatory receptors, as it does for m o d u l a t o r y receptors 27' then the net consequence might be a ' d a m p i n g ' of synaptic activity acting to prevent long-term changes in synaptic activity. A s a working hypothesis we propose that various forms of cortical plasticity result from the c o m b i n e d weight of inhibitory and excitatory synaptic activity, differentially regulated by their various r e c e p t o r populations during early stages of postnatal life. Thus neuronal plasticity will occur following changes in neuronal activity i f there is a net decrease in inhibitory r e c e p t o r n u m b e r and, perhaps, an increase in excitatory r e c e p t o r number. The p e r i o d in which such changes are the most p r o m i n e n t would then constitute the p e a k of the 'critical period'. It is worth noting in this regard that the p e a k for inducing LTP in rat visual cortex appears to be near 15 days postnatal 22, close to our o b s e r v e d m a x i m u m in G A B A A receptor down-regulation. In addition, the peaks for both LTP induction in cat visual cortex (21-34 days postnatal 15) as well as for m o n o c u l a r deprivation plasticity (near 30 days) 11 occur m o r e or less coincidentally with the m a x i m u m G A B A A r e c e p t o r down-regulation to altered neural activity r e p o r t e d here in our cat data. In regard to m o n o c u l a r deprivation however, it is clear that such effects are quite complex and may not be easily c o m p a r e d to cortical LTP since they involve several stages, one of which ,may be the loss of a fraction of the terminals representing the closed eye (see ref. 7). In this m o d e l we further speculate that as the direction of regulation begins to shift for the various receptors with age, the ' e n v e l o p e ' of regulation closes and the critical p e r i o d comes to an end. We note that the only way that cortical LTP can be routinely observed in adult animals involves the experimental reduction in functional G A B A A receptors with bicuculline 2.
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Such a m o d e l requires much new information before it can be accepted. We do not know that excitatory receptors show reciprocal regulation to the G A B A A receptors in cortex and it is likely naive to assume that they will do so in all cases. Studies in progress are directed at this issue. F u r t h e r m o r e , the underlying mechanisms leading to G A B A A r e c e p t o r regulation at any age, let alone the a g e - d e p e n d a n t regulation we have rep o r t e d here, are not completely understood. Some aspects of such regulation a p p e a r to be related to the activity of r e c e p t o r kinases and phosphatases, leading to changes in the n u m b e r of functional G A B A A receptors via d e p h o s p h o r y l a t i o n reactions 5. If such were the case, it would be a simple and attractive mechanism for neuronal regulation if similar kinase/phosphatase reactions had reciprocal effects and/or different concentration dependencies on excitatory receptors such as the A M P A and N M D A subtypes. F u r t h e r m o r e , differential age-dep e n d e n t regulation of inhibitory and excitatory receptors might be m e d i a t e d by a g e - d e p e n d e n t kinases and phosphatases. While much of the foregoing is highly speculative, we believe that the present quantitative data suggest novel ways in which cortical r e c e p t o r populations may be altered and, in consequence of which, how alterations in synaptic function m a y occur. Future studies, perhaps using electrophysiological techniques c o m b i n e d with receptor assays, may open new vistas to one of the most pressing problems in the neurosciences, i.e., the restoration of plasticity in the brain after the normal term of the critical period.
Acknowledgements. The present studies were supported grants from the Canadian Medical Research Council, the British Columbia Health Care Research Foundation and the Adamson Foundation to C.S.C.S. is a BC Health Care Research Scholar. B.A.S. is the recipient of an NSERC Summer Fellowship. We thank B.A. Pasqualotto and R.A. Lanius for assistance and Drs. M. Wilkinson, J. Simmons and E Van Huizen for helpful comments on the manuscript.
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