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Developmental expression of ClC-2 in the rat nervous system1
Developmental Brain Research 108 Ž1998. 307–318
Interactive report
Developmental expression of ClC-2 in the rat nervous system
1
Gerald H. Clayton a , Kevin J. Staley a , Christine L. Wilcox b, Geoffrey C. Owens c , Roderic L. Smith a,) a
c
Departments of Neurology and Pediatrics, UniÕersity of Colorado Health Sciences Center, DenÕer, CO 80262, USA b Department of Microbiology, Colorado State UniÕersity, Fort Collins, CO 80523, USA Department of Biochemistry, Biophysics, and Genetics, UniÕersity of Colorado Health Sciences Center, DenÕer, CO 80262, USA Accepted 25 December 1997
Abstract Regulation of expression of the voltage-gated chloride channel, ClC-2, was investigated during development and adult life in rat brain. RNase protection assays demonstrated a marked increase in levels of expression of ClC-2 in brain during early postnatal development which was also detected in adult brain. In situ hybridization of E15 and E18 rat brains demonstrated ClC-2 expression in deep brain nuclei and scattered cells within the neuroepithelial layers, but not in the regions of subventricular zone that primarily give rise to glial populations. By E18 all neurons within the emerging cortical plate and its equivalent in other areas of the CNS were heavily labeled. During the first postnatal week, ClC-2 was highly expressed in most neurons. By P7 a pattern of differential expression emerged with evidence of decreased expression of ClC-2 mRNA in many neuronal populations. In adult rat brain, ClC-2 was expressed at highest levels in large neurons as found within layer V of cortex, Ammon’s Horn of hippocampus, or mitral cells of the olfactory bulb and Purkinje cells within the cerebellum. Many smaller neurons within the diencephalon maintained significant levels of expression. A functional conductance was readily detected in hippocampal neurons during the first postnatal week, which had the same characteristic properties as the conductance observed in adult neurons. The observed expression and functional presence of ClC-2 suggest a widespread role in neuronal chloride homeostasis in early postnatal life, and demonstrated that cell specific shut-down resulted in the adult pattern of expression. q 1998 Elsevier Science B.V. All rights reserved. Keywords: Chloride channel; GABA; Depolarization; Ontogeny
1. Introduction The functional components of the gamma-amino butyric acid ŽGABA. neurotransmitter system are present during early development of the mammalian central nervous system ŽCNS. w16,20,25,41x. In the adult brain GABA is the principal neurotransmitter responsible for inhibition and elicits a hyperpolarizing response by activation of GABA A receptorrchloride channel complexes w21x. In contrast, GABA A activation in the embryonic brain mediates a striking depolarization that is sufficient to activate voltage-gated Ca2q channels w18,23x. The embryonic depolarizing response to GABA A activation is the result of high intra-neuronal concentrations of Cly, which is achieved by active accumulation via furosemide-sensitive transport w18x. During cortical development, neuronal intracellular Cly ) 1
Corresponding author. E-mail: [email protected] Published on the World Wide Web on 7 April 1998.
0165-3806r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 1 6 5 - 3 8 0 6 Ž 9 8 . 0 0 0 4 5 - 5
decreases significantly from embryonic to early postnatal life w24x, resulting in a shift from an excitatory to inhibitory GABA A response. During the first week of postnatal life, GABA A -mediated excitation is observed in hippocampal pyramidal neurons of CA3 before disappearing during the second postnatal week w4,5,7x. The role of changing Cly homeostatic mechanisms has been suggested as a significant factor in maturation of the GABA A response in both neocortex and hippocampus w18,19,21,24x. Developmental control of neuronal Cly homeostasis is significant for understanding the ontogeny of GABA A inhibition and age-related changes in neuronal homeostasis. Voltage-gated Cly channels have been shown to stabilize Cly concentrations in adult neurons w34,37x. Members of the voltage gated Cly channel family that are expressed at significant levels in brain include ClC-2 w40x, ClC-3 w12x, ClC-4 w42x, ClC-5 w31,39x, ClC-6 w6x, and ClC-7 w6x. Only ClC-2 has been demonstrated both to be expressed in neurons and to activate near the resting membrane poten-
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tial w34,37x. However, it remains possible that additional members of the ClC family of chloride channels may also contribute to this process. By stabilizing the equilibrium chloride concentration ŽE cl . near the resting membrane potential, ClC-2 or other chloride channels provides a mechanism for preventing neuronal Cly accumulation and GABA A -mediated depolarization. Therefore, the developmental pattern of ClC-2 expression is one of the potential molecular mechanisms that contributes to the reversal of the GABA A receptor-mediated response during development. To examine ClC-2 expression during development, we used both molecular and functional methods. The molecular methods allowed the expression of ClC-2 to be examined throughout brain and to identify regional changes in expression. These data provide a means of comparison with known time courses of development of neuronal chloride homeostasis. The functional studies allowed comparison of the properties of ClC-2 in selected populations of neurons at different stages of development.
tration of 5 m grml with either an anti-sense or sense-strand specific probe transcribed from a plasmid linearized with the appropriate restriction enzyme using either T3 or T7 DNA dependent RNA polymerase in the presence of a labeling mixture containing digoxigenin-UTP and UTP at a ratio of 0.54 ŽBoehringer–Mannheim.. The conditions of hybridization, RNase treatment and washing were as previously described w34x. Images of representative sections were captured using a COHU 4900 series CCD camera ŽCohu, San Diego, California. fitted to a Nikon Optiphot microscope ŽNikon, Melvile, New York. equipped with Hoffman optics. Digitized images were captured with a PowerMac 7100 computer ŽApple Computer, Cupertino, California. equipped with a video framegrabber card ŽLG-3, Scion, Frederick, Maryland. and image analysis software ŽImage 1.61, National Institutes of Health.. Images were then processed using the software package Photoshop 3.0.5 ŽAdobe Systems, Mountain View, California. and printed on a Tektronics Phaser IISDX dye-sub printer ŽTektronix, Wilsonville, Oregon..
2. Materials and methods
2.3. Ribonuclease protection analysis (RPA)
2.1. Tissue preparation
RPA was performed using total RNA obtained by extraction with Trizol ŽLife Technologies, Gaithersburg, MD. from the homogenized brains of embryonic ŽE15., postnatal ŽP0, P3, P7, P21. and adult animals. For analysis 10 m g of total RNA from each time point was hybridized with a 32 P labeled riboprobe 434 bp in length which protected a 312 bp fragment corresponding to bp 2391–2703 of ClC-2 w40x. This probe is a truncated version of one used for in situ hybridization studies of ClC-2 in adult rat brain w34x. The same probe utilized for in situ hybridization was also used in RNase protection to demonstrate equivalent results. Labeling and subsequent RNase digestion were performed using the Maxiscript and RPA II kits ŽAmbion, Austin, Texas. per the manufacturers instructions. RNA was simultaneously hybridized with a 32 P labeled probe which protected a 103 bp fragment of the constituitively expressed housekeeping gene cyclophilin ŽpTRI-Cyclophilin DNA template, Ambion.. This was done to provide a normalizing reference for subsequent analysis. Both probes were also hybridized with 10 m g of total yeast RNA as a control. Precipitated hybrids were then electrophoresed on a sequencing gel Ž6% Long Ranger; J.T.Baker, Phillipsburg, NJ. under denaturing conditions using standard methods w32x and scanned and analyzed using a Molecular Dynamics Phosphoimager ŽSunnyvale, CA..
All animals were treated in accordance with standards set forth by the author’s institution and the National Institutes of Health. Every effort was made to minimize pain or discomfort for the animals used in these experiments. Following Metofane Žmethoxyflurane, inhaled. overdose, embryonic rat brains were removed and immersion fixed in molecular biology grade HC fixative ŽAmresco, Solon, OH. for 24 h at room temperature, dehydrated and paraffin embedded by standard methods. Following overdose with Metofane or pentobarbitol, postnatal and adult rat brain was prepared by perfusion with molecular biology grade HC fixative followed by immersion fixation for several hours. The brains were then cut into 0.5 cm slices and immersion fixed overnight prior to dehydration and paraffin embedding. Ten m m sections were prepared from paraffin blocks and mounted on pretreated slides ŽSuperfrost plus, Fisher., and stored desiccated at y208 C until use. Immediately prior to use, the tissue sections were cleared of paraffin with xylene washes and rehydrated. 2.2. In situ hybridization histochemistry (ISHH) For the creation of probes for in situ hybridization cDNA clones derived from the 5X coding region of ClC-2 Žbp 42-277. were used as described previously w34x. These clones are postulated to contain sequences unique to this member of the voltage gated Cly channel family and should be species specific. Clones were subsequently used as templates for the transcription of digoxigenin labeled cRNA probes. In situ hybridization for detection of all species of mRNA was performed at a final probe concen-
2.4. Electrophysiology Whole cell recordings were obtained at 348C in rat hippocampal slices as previously described w37x. Where ClC-2 activation was investigated, Naq, Kq, and Ca2q y conductances, HCOy exchange, and cation-Cly co3 –Cl transport were blocked by using artificial cerebrospinal
G.H. Clayton et al.r DeÕelopmental Brain Research 108 (1998) 307–318
fluid ŽACSF. composed of 109 mM NaCl, 1.25 mM NaH 2 PO4 , 10 mM glucose, 20 mM tetraethylammonium chloride, 2.5 mM CsCl, 5 mM 4-aminopyridine, 4 mM MgCl 2 , 26 mM N-Ž2-hydroxyethyl.piperazine-N X-Ž2ethanesulfonic acid. ŽHEPES., and 0.5 mM furosemide. The pH was adjusted to 7.4 with NaOH, and the ACSF was saturated with 100% O 2 . Electrode solutions included 120 mM CsCl, 2 mM MgCl 2 , 7 mM NaCl, 2 mM QX314, 10 mM HEPES, 1 mM potassium ethylene glycol-bisŽbaminoethyl ether. N,N,N X ,N X-tetraacetic acid ŽEGTA., 4 mM potassium adenosine 5X-triphosphate, and 0.3 mM sodium guanosine 5X-triphosphate; pH 7.2. When the resting membrane potential ŽRMP. was measured, ACSF was composed of 126 mM NaCl, 2.5 mM KCl, 26 mM HEPES, 2 mM CaCl 2 , 2 mM MgCl 2 , 1.25 mM NaH 2 PO4 , 10 mM glucose, and 0.5 mM furosemide; pH 7.4; Kq replaced Csq in the electrode solution, and QX314 was not used. Leak subtraction for ClC-2 exploited the slow activation and inward rectification of this conductance: the clamp current within 50 ms of stepping to the test voltage was subtracted from the steady-state current Žafter 4 s at the test potential.. Normalized, leak-subtracted currents were fit to a Boltzman equation as described previously w37x. Because GABA A conductances and known voltage-dependent Cly conductances do not inwardly rectify, they do not contribute to this estimation of the ClC-2 conductance.
3. Results During early brain development, intracellular Cly concentrations are maintained in neurons and neuronal precursors at levels that are much higher than the passive equilibrium concentration ŽE cl . predicted by the resting membrane potential and at this stage GABA A activation results in strong depolarization w18,24x. Inwardly directed cationCly cotransporters are suggested as the likely mechanism for Cly accumulation. With maturity, neuronal Cly levels change significantly such that GABA A activation results in a hyperpolarizing Cly influx. In earlier studies it was shown that de novo expression of ClC-2 in embryonic DRG neurons via viral vector counteracted the ability of the neuron to accumulate Cly thereby decreasing its concentration to that predicted by E cl w36x. These results suggest that ClC-2 expression may contribute to the development of a mature pattern of Cly homeostasis. To understand the contribution of ClC-2 expression to development of mature neuronal Cly homeostasis, both molecular detection of expression of ClC-2 and functional studies were performed. 3.1. ClC-2 mRNA expression is deÕelopmentally regulated in rat brain To determine the developmental time course of ClC-2 mRNA expression, ribonuclease protection analysis was
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Fig. 1. Ribonuclease protection analysis of ClC-2 expression in developing and adult rat brain. RPA analysis was performed by hybridizing 10 m g of total RNA with 32 P-labeled riboprobes which protected unique regions of ClC-2 ŽA. and the cellular housekeeping gene cyclophilin ŽB.. In lane 1 hybridization with total yeast RNA was performed as a control. Lanes 2–7 demonstrate results of hybridization with total RNA from E15, P0, P3, P7, P21, and adult rat brains. A. ClC-2 expression increases from a low at E15 to a peak at P3 which is maintained through adulthood. Note the lack of expression in yeast total RNA. B. Expression of the cellular housekeeping gene cyclophilin is not present in yeast and does not vary during development.
performed on total RNA obtained from rat brains ranging in age from E15 to adult. As shown in Fig. 1A, ClC-2 mRNA was detected at moderate levels in E15 brain Ž38% of adult., reaching peak levels by day P3, and was maintained at similar levels throughout postnatal development and into adulthood. Simultaneous hybridization of total brain RNA with probes for ClC-2 and cyclophilin ŽFig. 1B. detected unique protected fragments of expected sizes Žsee materials and methods.. Levels of cyclophilin, a cellular housekeeping gene, in the nuclease protection assay were used to confirm that equivalent amounts of RNA were used in each hybridization reaction. The 5X coding region probe used for ISHH described above was also used for RPA analysis Ž211 bp protected fragment. with identical results Ždata not shown.. These results indicate that significant levels of ClC-2 were expressed during the appearance of cortical neurons. 3.2. Localization of ClC-2 expression in deÕeloping brain The use of whole brain RNA to measure ClC-2 expression can obscure potentially important regional differences in ClC-2 expression. The level of ClC-2 expression varies greatly in neuronal populations in the adult brain w34x. In situ hybridization methods were used to examine the pattern of ClC-2 expression during development and to determine if significant differences exist in abundance of ClC-2 expression. 3.2.1. Cortex Embryonic brains of E15 rats demonstrated ClC-2 in situ hybridization signal in a small percentage of the
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Fig. 2. ClC-2 mRNA is expressed in neuronal precursors in prenatal rat brain. The distribution of ClC-2 mRNA was examined in embryonic day 15 and 18 rat brain using in situ hybridization methods. ŽA. E15 brain showing the cortical and striatal germinal matrices Ži.e. ventricular zone, VZ., surrounding the lateral ventricle. Scattered ClC-2 expressing cells can be seen within these regions in addition to limited signal within the beginnings of the cortical plate ŽCP.. ŽB. E18 brain demonstrates strongly labeled cells scattered throughout the ventricular zone. Diffuse staining was seen within the cortical plate just below the marginal zone ŽMZ, arrow. and a thin, lightly labeled subplate layer ŽSP, arrow. at the inner border of the CP. ŽC. Higher power view of E15 brain depicted in ‘A’ clearly showing scattered labeling in the VZ Žbracketed by arrows. as well as labeled cells in the newly formed CP Žarrowheads.. ŽD. Higher power view of E18 brain depicted in ‘B’ showing scattered labeling within the VZ with an accumulation of labeled cells demonstrated at its outer border Žarrowheads.. ŽE. E15 hippocampus showing ClC-2 label within the developing CA1 region Žarrow.. ŽF. ClC-2 expression in E18 hippocampus. The CA1 region Žarrow. is heavily labeled while dentate gyrus ŽDG. is relatively devoid of signal. Scale bar in A s 200 m m and applies to B, E, and F. Scale bar in C s 100 m m and also applies to D.
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Fig. 3. Expression of ClC-2 mRNA in cerebral cortex is developmentally regulated. ClC-2 mRNA expression was examined by in situ hybridization in coronal sections through cortex near the lateral ventricle from postnatal ages P0-adult. ŽA. P0: ClC-2 hybridization signal was significantly increased above embryonic levels. Heaviest labeling is seen within a narrow band within the CP Žasterisk., the supraventricular subplate region Žarrowhead., and a now very limited germinative VZ Žarrow.. ŽB. P0: Control hybridization performed with a sense-strand probe demonstrates absence of signal. ŽC. P7: All cortical layers are present. Heaviest labeling can be seen within a thin CP band at the border of the marginal zone Žasterisk., within layer V neurons, and in the subplate region Žarrowhead.. ŽD. P14: ClC-2 hybridization signal is evident in most cells with heaviest label in layer V and in the now limited subplate layer Žarrowhead.. ŽE. Adult: Most neurons contain some ClC-2 signal with the most prominent label still evident within layer V, however the subplate layer is no longer in evidence. Scale bars in all figuress 200 m m.
Fig. 4. Developmental changes in ClC-2 mRNA expression in caudoputamen ŽCPU.. Examination of ClC-2 hybridization within coronal sections through the CPU from ŽA. embryonic day 18 ŽE18., and ŽB. adult rat brains. These results demonstrate that the widespread embryonic expression of ClC-2 mRNA becomes markedly restricted to a limited population of neurons in the adult. The scale bar in A is 200 m m and also applies to panel B.
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densely packed cellular profiles of the proliferative neuroepithelium or ventricular zone ŽVZ. of developing cortex ŽFig. 2.. The majority of cells within this region did not demonstrate significant clc-2 staining consistent with the published observations of chloride accumulation and GABA A -mediated depolarization w18x. A similar pattern of clc-2 expression was observed in E18 brains within the VZ, although strongly positive profiles were apparent at the boundary of the ventricular and subventricular zones ŽFig. 2D.. Only rare ClC-2 positive profiles were detected in the subventricular zone, which largely gives rise to glial populations Žfor review see w10x.. The absence of ClC-2 expression in the subventricular zone is consistent with the lack of ClC-2 expression in glial regions in adult rat brain w34x. In contrast, significant levels of expression were present in the cortical plate and subplate neurons ŽFig. 2A, B, and C.. Similarly, ClC-2 hybridization signal was present in the region of the cortical plate that gives rise to the CA1 subfield but was not expressed to detectable levels in the secondary germinal cell mass that generates dentate
gyrus ŽFig. 2E and F.. These results suggest that ClC-2 expression is confined to cells committed to neuronal differentiation. From P0–P7 increasing signal was detected in virtually all cortical layers including remaining neurons of VIb ŽFig. 3A and C.. By P14, all cortical layers seen in the adult are distinct and express ClC-2 ŽFig. 3D.. However, a decline in the intensity of the ClC-2 signal in some layers was evident with the highest intensity of signal in layer V. ClC-2 signal levels declined further with maturity ŽFig. 3E. however, expression remained high within layer V as previously reported w34x. Restriction of cortical ClC-2 to larger output neurons is a theme repeated in other areas however, the timing and extent of change in expression was different in different regions of brain. 3.2.2. Caudoputamen The pattern of expression of ClC-2 observed in developing cortex suggested that expression of ClC-2 during development was related to the timing of neuronal differenti-
Fig. 5. In olfactory bulb, ClC-2 expression becomes restricted primarily to mitral cells during postnatal development. ClC-2 hybridization signal within coronal andror sagittal sections through the rat olfactory bulb ŽOB.. At postnatal day 0 ŽP0, A and D. proliferating andror migrating cells expressed ClC-2 mRNA throughout the OB with the exception of the olfactory glomeruli ŽGL.. Heaviest labeling is seen within the very broad mitral cell layer ŽMCL.. By postnatal day 7 ŽP7, B and E. labeling is much more restricted. However, the mitral cell layer is still thickened with prominent labeling just under and surrounding the glomeruli Žasterisk.. The unlabeled olfactory nerve layer ŽONL. can be seen just external to the glomeruli. In the adult ŽC and F. ClC-2 labeling is restricted to a 1 or 2 cell thick mitral cell layer with some labeling of cells seen just internal to the glomeruli. Scale bars in A, B, and C s 200 m m, D, E, and F s 100 m m.
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ation. The differentiating striatal and pallidal regions were easily recognizable at E18 ŽFig. 4A. with most of the cells within these structures demonstrating moderate to high levels of ClC-2 hybridization signal. Expression of ClC-2 was significantly reduced in most neurons of striatum by early postnatal life, although a limited population retained expression until adulthood ŽFig. 4B.. These data provide clear evidence of selective down-regulation of expression as the major mechanism for establishing postnatal and adult patterns of ClC-2 expression w34x. 3.2.3. Olfactory bulb In structures with significant postnatal neurogenesis and migration, such as olfactory bulb and cerebellum, ClC-2 expression was delayed. ClC-2 expression in the olfactory
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bulb was first observed on embryonic day 18 and restricted to the developing mitral cell layer Ždata not shown. whose resident neurons are the earliest born w1x. By P0 however, cells within all areas of the bulb exhibited heavy labeling with the exception of the glomeruli ŽFig. 5A and D.. The most intense signal was associated with the mitral cell layer with numerous smaller, well-labeled cells in evidence external to and internal to this layer. Between P7 and P14 ŽFig. 5B and E. the pattern of ClC-2 expression became more restricted, primarily limited to mitral cells in the adult ŽFig. 5C and F.. 3.2.4. Cerebellum By E18 ClC-2 hybridization signal was first detected in the neurons of the deep nuclei of cerebellum Ždata not
Fig. 6. Developmental changes in ClC-2 expression within the cerebellum. In the postnatally developing cerebellum ClC-2 hybridization signal occurred within the majority of cells but with maturity became restricted to Purkinje neurons. At postnatal day 3 ŽP3, A and C. heaviest label can be seen within the subpial external germinal layer ŽEGL., the multi-cell thick, prenatally formed Purkinje cell layer underneath ŽP., and cells within the deep nuclei Žasterisk.. The unlabeled band between the EGL and the Purkinje cell layer is the beginning of the molecular layer ŽM.. Post migratory granule cells ŽG. lie underneath the Purkinje cell layer and can be seen to be labeled as well. Other heavily labeled cells Žlikely in transit. can also be identified. In the adult ŽB and D., ClC-2 mRNA becomes restricted to the monolayer of Purkinje cells and what appears to be some externally displaced smaller cells within the molecular layer. Scale bars in A and B s 200 m m and scale bars in C and D s 100 m m.
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shown.. These neurons remained heavily labeled in the adult animal. Between P3 and P7 cells within the external germinal layer and Purkinje cell layer were heavily labeled ŽFig. 6A and C.. By P21 the adult pattern of ClC-2 labeling developed with the most intense signal in Purkinje cells, with some occasional labeling detected in large cells within the granular layer and a few scattered cells in the molecular layer ŽFig. 6B and D.. This pattern remained in all subsequent ages examined. 3.2.5. Hippocampus From embryonic ŽFig. 2E and F. to adult animals ŽFig. 7. pyramidal neurons of Ammon’s Horn Žfields CA1-4. demonstrated marked ClC-2 signal. In dentate gyrus, ClC-2 was not expressed prenatally nor to any significant degree in the adult. However, a thin layer of cells in the region of the earliest maturing neurons of the dentate gyrus transiently expressed ClC-2 hybridization signal during the first weeks of postnatal development ŽFig. 7C.. By P14 the
expression of ClC-2 was restricted to Ammon’s Horn and had assumed an adult-like distribution as described in previous work w34x ŽFig. 7B, D, and F.. 3.3. Functional expression of ClC-2 In previous studies we have correlated ClC-2 expression with the presence of a characteristic inwardly rectifying conductance in hippocampus of brain slices prepared from adult brains w35,37x. The presence of high levels of ClC-2 in pyramidal neurons of hippocampus during the first week of postnatal life would not be predicted if the GABAmediated depolarization that has been reported for CA3 w4,5,7x is due to intraneuronal chloride accumulation. An inwardly rectifying chloride conductance was present in P3–4 CA1 pyramidal cells ŽFig. 8A. consistent with the local expression of ClC-2 described above. This conductance was smaller than in adult pyramidal cells, but still large enough to prevent substantial chloride accumulation
Fig. 7. Developmental changes in ClC-2 mRNA expression in hippocampus. As determined by in situ hybridization, ClC-2 was expressed within most neuronal populations of the hippocampus by P0. However, expression was restricted to cells within Ammon’s Horn with maturity. In panel A the extent of ClC-2 expression can be seen in the P0 hippocampus. Heavy labeling can be identified within Ammon’s Horn ŽCA1 and CA3., the hilar region ŽH. and dentate gyrus ŽDG, arrows.. In panel C Žmagnified view of panel A. the extent of labeling is more apparent and migrating granule cells can be seen forming a line underneath CA1 Žarrow.. Positively labeled cell can also be identified along the hippocampal fissure Žasterisk.. Panel B Žadult, 9 months. demonstrates the extent to which ClC-2 expression becomes limited with maturity. Panel D Žmagnified view of panel B. shows the loss of ClC-2 expression within dentate granule cells. Note that expression of ClC-2 within choroid epithelia is evident by P0, and is maintained into adulthood Žasterisk in panels A and B.. The scale bars in A and B s 200 m m; the scale bars in C and D s 100 m m.
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Fig. 8. Electrophysiological analysis of ClC-2-mediated currents in developing rat brain. ŽA. The maximal conductance calculated for an inwardly rectifying chloride current in CA1 pyramidal cells from P3 Ž n s 8. and adult Ž n s 5. rats. The ClC-2 conductance was calculated as previously described w36,37x. ŽB. The ClC-2 conductance was large enough to prevent chloride accumulation in P3 neurons. The resting membrane potential of a P3 pyramidal cell was gradually depolarized due to chloride efflux via ClC-2 when recorded with a 135 mM chloride electrode solution Ž n s 2; w37x.. In contrast, the RMP of P3 pyramidal cells increased or was unchanged in 6 recordings with electrode solutions containing 4 mM chloride.
in pyramidal cells recorded with high-chloride electrodes ŽFig. 8B..
4. Discussion In this study, RPA analysis demonstrated a 2–3 fold increase in ClC-2 mRNA levels between E15 and early postnatal life. The levels of mRNA measured in total brain RNA were likely to under-represent the increases in neurons, since glial components contribute increasingly to brain mass later in development. In most regions of brain examined ClC-2 expression was initially diffuse, but later became restricted to specific neuronal populations. The in situ hybridization methods may not allow direct comparison of expression at different developmental stages; however, striking differential expression emerged as brain structures matured. In late developing structures, such as cerebellum and olfactory bulb, expression of ClC-2 was delayed. Large projection neurons, including mitral cells of the olfactory bulb, Purkinje cells of the cerebellum, pyramidal neurons of cortex and hippocampus, were the primary neurons within their respective structures that main-
tained high levels of ClC-2 expression. Virtually all neurons demonstrated expression early in development, although down-regulation of mRNA levels was evident in striatum by early postnatal life. The timing and localization of increasing ClC-2 expression was consistent with the transition from high intracellular Cly to levels approaching a passive distribution that has been reported during the transition from late embryonic to early postnatal life in cerebral cortex w24x. The ventricular zone of developing cortex gives rise to cells committed to differentiate into cortical neurons. GABA A agonists applied to cell populations from ventricular zone depolarize them to a degree sufficient to activate voltage-sensitive Ca2q channels w18,23x. Consistent with this observation, the majority of cell profiles in this region were negative for ClC-2 expression ŽFig. 2., since expression of ClC-2 would be expected to prevent Cly accumulation which is necessary for GABA A -mediated depolarization. The significance of the observed expression of ClC-2 in a subpopulation of ventricular zone cells is unknown. However, this could provide a mechanism for altering the response to GABA signaling in selected cortical precursors thereby preventing the GABA induced depolarization nor-
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mally observed in this region. Neurotransmitter induced activity can alter the migratory behavior of cortical precursors w2,3,14,43x as well as affect DNA synthesis w18x likely via Ca2q-mediated mechanisms. In other regions of the brain, such as the cerebellum and olfactory bulb, migrating populations of neuronal precursors also demonstrate ClC-2 signal ŽFig. 6A and D.. In contrast, regions that primarily give rise to glia such as the cortical subventricular zone did not demonstrate hybridization signal with the exception of rare profiles which may represent neuronal precursors in transit to cortical plate or the small percentage subventricular precursor cells that become neurons Žsee w11x for review.. Throughout development, high level expression of ClC-2 was confined to neuronal regions, although it remains possible that much lower levels of expression may occur in some glial populations that are below the limit of detection of in situ techniques. The ClC-2 mRNA expression observed during the first postnatal week of life was correlated with presence of a functional conductance with the same properties as observed following viral-mediated gene transfer to non-ClC-2 expressing neurons and observed in adult hippocampal pyramidal cells w35–37x. In pyramidal neurons of hippocampus, GABA A -mediated depolarization continues after the appearance of significant levels of ClC-2 w4,5,7x. Several possible mechanisms could account for this apparent contradiction and contribute to GABA A -mediated depolarization after ClC-2 expression. First, ClC-2 is subject to regulation by stimuli that include changes in extracellular pH, exogenous Zn2q, and activators of protein kinase C w36,37x, and may not be active under the conditions utilized for these studies. Secondly, flux of bicarbonate via the GABA A -gated anion channel can produce a significant depolarization after collapse of Cly gradients w38x. Regardless of the specific explanation for the GABA A -mediated depolarization, the experimental evidence clearly confirms the presence of ClC-2, which should oppose neuronal accumulation of Cly. The functional role of ClC-2 may vary significantly throughout development. In addition to playing a role in the prevention of Cly accumulation, this channel has also been proposed to serve as a volume-regulator in non-neuronal tissues. The characterization of ClC-2 expressed in Xenopus oocytes demonstrates activation in response to extracellular hypotonic solutions w9x. Activation of ClC-2 and swelling activated Kq channels by hypotonic stimuli provides a potential mechanism for regulatory volume decrease ŽRVD. mediated by the outward movement of Cly and Kq w9,26,28,30,40x. In adult neurons, ClC-2 activation by intracellular Cly concentration is a significant feature of regulation w34,36,37x. At early stages of development, ClC-2 may have a more significant role in mediating volume-regulation in neuronal precursors than in the mature neurons, where control of intracellular Cly concentration is a significant effect. Functional studies of ClC-2 activation demonstrated a smaller absolute conduc-
tance activated by high intracellular Cly in P3 hippocampus; however, even at this stage of development ClC-2 was sufficient to strongly stabilize intracellular Cly concentration near E Cl . Control of ClC-2 by separate regulatory mechanisms may allow expression of ClC-2 in a sub-population of neuronal precursors that maintain high intracellular Cly w18,24x. In adult brain transmembrane neuronal Cly gradients are maintained at values that are near or below the resting membrane potential as a necessary condition for GABA A receptor-mediated postsynaptic inhibition. This transmembrane chloride gradient is not favorable for regulatory volume decrease mediated by ClC-2 activation and subsequent chloride efflux. An alternative function for ClC-2 is suggested by the varied experimental response to chloride loading by neurons that differentially express this channel w34,37x. In adult hippocampus the dentate gyrus granular neurons, which lack ClC-2, can be readily loaded with Cly in whole cell clamp studies, while pyramidal neurons resist loading with Cly because of the activation of ClC-2 w37x. In neurons that express ClC-2, activation by increasing intracellular Cly has been demonstrated under isotonic conditions w37x. In embryonic DRG neurons, the de novo expression of ClC-2 by virus vector-mediated gene transfer resulted in a significant shift in neuronal Cly concentrations prior to any change in cell volume w36x. Neurons have special requirements to regulate intracellular Cly separate from cell volume, since the transmembrane Cly concentration gradient is a primary determinant of GABA A -mediated signal transduction. The differential expression of ClC-2 in neurons during maturation is a striking feature that has not been reported for other voltage-gated-chloride channels w12x. These findings indicate that components of Cly homeostatic control change over a significant period of life and may contribute to homeostatic changes in adult or aging brain. These observations suggest that certain classes of neurons have retained ClC-2 as a high capacity system to prevent Cly loading from occurring. The mechanisms that control ClC-2 expression are unknown; however, ClC-2 expression is precisely regulated in tissues such as lung with marked shut-down in expression within 24 h of birth w22x. In contrast, the changes in ClC-2 expression observed in rat brain extend over a long time period and involve both cell-specific increases and decreases in expression during development. The net effect is that the total abundance of ClC-2 expression is little changed from early postnatal to adult life in rat brain. The ontogeny of neuronal Cly homeostasis involves not only anion channels but also involves cation-Cly co-transport and anion-exchange mechanisms. The cation y Cly co-transporters, which couple movement of Cly to cationic gradients, are likely to be significant both early in development and during post-natal life w18,24x. The initially depolarizing actions of GABA A agonists on neuronal precursors are dependent on active accumulation of Cly against
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the concentration gradient by a furosemide-inhibitable mechanism suggesting a role for inwardly directed cationCly cotransport w18,24x. This process likely requires unique inwardly directed cation-Cly cotransporters. In previous studies we have demonstrated that de novo expression of ClC-2 using a viral vector is sufficient to stabilize neuronal Cly concentrations near E Cl in cultured embryonic DRG neurons which normally actively accumulate Cly and depolarize in response to GABA A activation w36x. During the period when increased ClC-2 expression was observed, Cly concentrations in neurons approach concentrations predicted by a passive distribution, suggesting a role for ClC-2 during this stage of development. Postnatally, the intraneuronal concentration of Cly decreases until an adult pattern is attained with Cly maintained at concentrations less than predicted by E Cl w24x suggesting a role for outwardly directed transport mechanisms such as the recently described Kq dependent cation-Cly cotransporters, KCC-1 and KCC-2 w8,27x. Anion exchange mechanisms may also significantly contribute to the developmental changes in Cly homeostasis within the nervous system. These proteins have been identified in neuronal and nonneuronal cells, have been shown to be developmentally regulated in some populations, and have been suggested to play a role in neural induction w13,15,17,29,33x. Therefore, changes in ClC-2 expression are likely to be one component that contributes to developmental alterations in neuronal chloride homeostasis and the reversal of GABA A mediated depolarization.
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Acknowledgements This work is supported by grants from the NIH including a training grant NS 0732 to GHC, NS 34360 to KJS, and a grant from the March of Dimes 1-FY- 0454 to RLS.
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References w1x S.A. Bayer, J. Altman, Neurogenesis and neuronal migration, in: G. Paxinos ŽEd.., The Rat Nervous System, Academic Press, Sydney, 1995, pp. 1041-1078. w2x T.N. Behar, Y. Li, H.T. Tran, W. Ma, V. Dunlap, C. Scott, J.L. Barker, GABA stimulates chemotaxis and chemokinesis of embryonic cortical neurons via calcium-dependent mechanisms, J. Neurosci. 16 Ž1996. 1808–1818. w3x T.N. Behar, A.E. Schaffner, C.A. Colton, R. Somogyi, Z. Olah, C. Lehel, J.L. Barker, GABA-induced chemokinesis and NGF-induced chemotaxis of embryonic spinal cord neurons, J. Neurosci. 14 Ž1994. 29–38. w4x Y. Ben-Ari, E. Cherubini, R. Corradetti, J.L. Gaiarsa, Giant synaptic potentials in immature rat CA3 hippocampal neurons, J. Physiol. 416 Ž1989. 303–325. w5x Y. Ben-Ari, V. Tseeb, D. Raggozzino, R. Khazipov, J.L. Gaiarsa, g-aminobutyric acid ŽGABA.: a fast excitatory transmitter which may regulate the development of hippocampal neurones in early postnatal life, Prog. Br. Res. 102 Ž1994. 261–273. w6x S. Brandt, T.J. Jentsch, ClC-6 and ClC-7 are two novel broadly
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w29x K.M. Raley-Susman, R.M. Sapolsky, R.R. Kopito, ClyrHCOy 3 exchange function differs in adult and fetal rat hippocampal neurons, Br. Res. 614 Ž1993. 308–314. w30x H.A. Sackin, Stretch-activated potassium channels in renal proximal tubule, Am. J. Physiol. 253 Ž1987. F1253–F1262. w31x H. Sakamoto, M. Kawasaki, S. Uchida, S. Sasaki, F. Marumo, Identification of a new outwardly rectifying Cly channel that belongs to a subfamily of the ClC Cly channels, J. Biol. Chem. 271 Ž1996. 10210–10216. w32x J. Sambrock, E.F. Fritich, T. Maniatis, Molecular cloning: A laboratory Manual, Cold Spring Harbor Press, New York, 1989. w33x A.K. Sater, J.M. Alderton, R.A. Steinhardy, An increase in intracellular pH during neural induction in Xenopus, Development. 120 Ž1994. 433–442. w34x R.L. Smith, G.H. Clayton, C.L. Wilcox, K.W. Escudero, K.J. Staley, Differential expression of an inwardly rectifying chloride conductance in rat brain neurons: A potential mechanism for cell-specific modulation of postsynaptic inhibition, J. Neurosci. 15 Ž1995. 4057– 4067. w35x R.L. Smith, A.I. Geller, K. Escudero, C.L. Wilcox, Long-term expression in neuronal cultures of E.coli ß-galactosidase under the control of promoters from herpes simplex virus immediate early genes 1, 3, 4r5, J. Virol. 69 Ž1995. 4593–4599. w36x K. Staley, R. Smith, J. Schaack, C. Wilcox, T. Jentsch, Alteration of GABA A receptor function following gene transfer of the ClC-2 chloride channel, Neuron 17 Ž1996. 543–551.
w37x K.J. Staley, The role of an inwardly rectifying chloride conductance in postsynaptic inhibition, J. Neurophysiol. 72 Ž1994. 273–284. w38x K.J. Staley, B.L. Soldo, W.R. Proctor, Ionic mechanisms of neuronal excitation by inhibitory GABA A receptors, Science 269 Ž1995. 977–981. w39x K. Steinmeyer, B. Schwappach, M. Bens, A. Vandewalle, T.J. Jentsch, Cloning and functional expression of rat ClC-5, a chloride channel related to kidney disease, J. Biol. Chem. 270 Ž1995. 31172– 31177. w40x A. Thiemann, S. Grunder, M. Pusch, T.J. Jentsch, A chloride channel widely expressed in epithelial and non-epithelial cells, Nature 356 Ž1992. 57–60. w41x C.G. Van Eden, L. Mrzljak, P. Voorn, H.B.M. Uylings, Prenatal development of GABA-ergic neurons in the neocortex of the rat, J. Comp. Neurol. 289 Ž1989. 213–227. w42x M.A. van Slegtenhorst, M.T. Bassl, G. Borsani, M.C. Wapenaar, G.B. Ferrero, L. de Concillis, E.L. Rugarli, A. Grillo, B. Franco, H.Y. Zoghbi, A. Ballabio, A gene from the Xp22.3 region shares homology with voltage-gated chloride channels, Hum. Mol. Gen. 3 Ž1994. 547–552. w43x R. Yuste, L. Katz, Control of postsynaptic Caqq influx in developing neocortex by excitatory and inhibitory neurotransmitters, Neuron 6 Ž1991. 333–344.
Interactive report
Developmental expression of ClC-2 in the rat nervous system
1
Gerald H. Clayton a , Kevin J. Staley a , Christine L. Wilcox b, Geoffrey C. Owens c , Roderic L. Smith a,) a
c
Departments of Neurology and Pediatrics, UniÕersity of Colorado Health Sciences Center, DenÕer, CO 80262, USA b Department of Microbiology, Colorado State UniÕersity, Fort Collins, CO 80523, USA Department of Biochemistry, Biophysics, and Genetics, UniÕersity of Colorado Health Sciences Center, DenÕer, CO 80262, USA Accepted 25 December 1997
Abstract Regulation of expression of the voltage-gated chloride channel, ClC-2, was investigated during development and adult life in rat brain. RNase protection assays demonstrated a marked increase in levels of expression of ClC-2 in brain during early postnatal development which was also detected in adult brain. In situ hybridization of E15 and E18 rat brains demonstrated ClC-2 expression in deep brain nuclei and scattered cells within the neuroepithelial layers, but not in the regions of subventricular zone that primarily give rise to glial populations. By E18 all neurons within the emerging cortical plate and its equivalent in other areas of the CNS were heavily labeled. During the first postnatal week, ClC-2 was highly expressed in most neurons. By P7 a pattern of differential expression emerged with evidence of decreased expression of ClC-2 mRNA in many neuronal populations. In adult rat brain, ClC-2 was expressed at highest levels in large neurons as found within layer V of cortex, Ammon’s Horn of hippocampus, or mitral cells of the olfactory bulb and Purkinje cells within the cerebellum. Many smaller neurons within the diencephalon maintained significant levels of expression. A functional conductance was readily detected in hippocampal neurons during the first postnatal week, which had the same characteristic properties as the conductance observed in adult neurons. The observed expression and functional presence of ClC-2 suggest a widespread role in neuronal chloride homeostasis in early postnatal life, and demonstrated that cell specific shut-down resulted in the adult pattern of expression. q 1998 Elsevier Science B.V. All rights reserved. Keywords: Chloride channel; GABA; Depolarization; Ontogeny
1. Introduction The functional components of the gamma-amino butyric acid ŽGABA. neurotransmitter system are present during early development of the mammalian central nervous system ŽCNS. w16,20,25,41x. In the adult brain GABA is the principal neurotransmitter responsible for inhibition and elicits a hyperpolarizing response by activation of GABA A receptorrchloride channel complexes w21x. In contrast, GABA A activation in the embryonic brain mediates a striking depolarization that is sufficient to activate voltage-gated Ca2q channels w18,23x. The embryonic depolarizing response to GABA A activation is the result of high intra-neuronal concentrations of Cly, which is achieved by active accumulation via furosemide-sensitive transport w18x. During cortical development, neuronal intracellular Cly ) 1
Corresponding author. E-mail: [email protected] Published on the World Wide Web on 7 April 1998.
0165-3806r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 1 6 5 - 3 8 0 6 Ž 9 8 . 0 0 0 4 5 - 5
decreases significantly from embryonic to early postnatal life w24x, resulting in a shift from an excitatory to inhibitory GABA A response. During the first week of postnatal life, GABA A -mediated excitation is observed in hippocampal pyramidal neurons of CA3 before disappearing during the second postnatal week w4,5,7x. The role of changing Cly homeostatic mechanisms has been suggested as a significant factor in maturation of the GABA A response in both neocortex and hippocampus w18,19,21,24x. Developmental control of neuronal Cly homeostasis is significant for understanding the ontogeny of GABA A inhibition and age-related changes in neuronal homeostasis. Voltage-gated Cly channels have been shown to stabilize Cly concentrations in adult neurons w34,37x. Members of the voltage gated Cly channel family that are expressed at significant levels in brain include ClC-2 w40x, ClC-3 w12x, ClC-4 w42x, ClC-5 w31,39x, ClC-6 w6x, and ClC-7 w6x. Only ClC-2 has been demonstrated both to be expressed in neurons and to activate near the resting membrane poten-
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tial w34,37x. However, it remains possible that additional members of the ClC family of chloride channels may also contribute to this process. By stabilizing the equilibrium chloride concentration ŽE cl . near the resting membrane potential, ClC-2 or other chloride channels provides a mechanism for preventing neuronal Cly accumulation and GABA A -mediated depolarization. Therefore, the developmental pattern of ClC-2 expression is one of the potential molecular mechanisms that contributes to the reversal of the GABA A receptor-mediated response during development. To examine ClC-2 expression during development, we used both molecular and functional methods. The molecular methods allowed the expression of ClC-2 to be examined throughout brain and to identify regional changes in expression. These data provide a means of comparison with known time courses of development of neuronal chloride homeostasis. The functional studies allowed comparison of the properties of ClC-2 in selected populations of neurons at different stages of development.
tration of 5 m grml with either an anti-sense or sense-strand specific probe transcribed from a plasmid linearized with the appropriate restriction enzyme using either T3 or T7 DNA dependent RNA polymerase in the presence of a labeling mixture containing digoxigenin-UTP and UTP at a ratio of 0.54 ŽBoehringer–Mannheim.. The conditions of hybridization, RNase treatment and washing were as previously described w34x. Images of representative sections were captured using a COHU 4900 series CCD camera ŽCohu, San Diego, California. fitted to a Nikon Optiphot microscope ŽNikon, Melvile, New York. equipped with Hoffman optics. Digitized images were captured with a PowerMac 7100 computer ŽApple Computer, Cupertino, California. equipped with a video framegrabber card ŽLG-3, Scion, Frederick, Maryland. and image analysis software ŽImage 1.61, National Institutes of Health.. Images were then processed using the software package Photoshop 3.0.5 ŽAdobe Systems, Mountain View, California. and printed on a Tektronics Phaser IISDX dye-sub printer ŽTektronix, Wilsonville, Oregon..
2. Materials and methods
2.3. Ribonuclease protection analysis (RPA)
2.1. Tissue preparation
RPA was performed using total RNA obtained by extraction with Trizol ŽLife Technologies, Gaithersburg, MD. from the homogenized brains of embryonic ŽE15., postnatal ŽP0, P3, P7, P21. and adult animals. For analysis 10 m g of total RNA from each time point was hybridized with a 32 P labeled riboprobe 434 bp in length which protected a 312 bp fragment corresponding to bp 2391–2703 of ClC-2 w40x. This probe is a truncated version of one used for in situ hybridization studies of ClC-2 in adult rat brain w34x. The same probe utilized for in situ hybridization was also used in RNase protection to demonstrate equivalent results. Labeling and subsequent RNase digestion were performed using the Maxiscript and RPA II kits ŽAmbion, Austin, Texas. per the manufacturers instructions. RNA was simultaneously hybridized with a 32 P labeled probe which protected a 103 bp fragment of the constituitively expressed housekeeping gene cyclophilin ŽpTRI-Cyclophilin DNA template, Ambion.. This was done to provide a normalizing reference for subsequent analysis. Both probes were also hybridized with 10 m g of total yeast RNA as a control. Precipitated hybrids were then electrophoresed on a sequencing gel Ž6% Long Ranger; J.T.Baker, Phillipsburg, NJ. under denaturing conditions using standard methods w32x and scanned and analyzed using a Molecular Dynamics Phosphoimager ŽSunnyvale, CA..
All animals were treated in accordance with standards set forth by the author’s institution and the National Institutes of Health. Every effort was made to minimize pain or discomfort for the animals used in these experiments. Following Metofane Žmethoxyflurane, inhaled. overdose, embryonic rat brains were removed and immersion fixed in molecular biology grade HC fixative ŽAmresco, Solon, OH. for 24 h at room temperature, dehydrated and paraffin embedded by standard methods. Following overdose with Metofane or pentobarbitol, postnatal and adult rat brain was prepared by perfusion with molecular biology grade HC fixative followed by immersion fixation for several hours. The brains were then cut into 0.5 cm slices and immersion fixed overnight prior to dehydration and paraffin embedding. Ten m m sections were prepared from paraffin blocks and mounted on pretreated slides ŽSuperfrost plus, Fisher., and stored desiccated at y208 C until use. Immediately prior to use, the tissue sections were cleared of paraffin with xylene washes and rehydrated. 2.2. In situ hybridization histochemistry (ISHH) For the creation of probes for in situ hybridization cDNA clones derived from the 5X coding region of ClC-2 Žbp 42-277. were used as described previously w34x. These clones are postulated to contain sequences unique to this member of the voltage gated Cly channel family and should be species specific. Clones were subsequently used as templates for the transcription of digoxigenin labeled cRNA probes. In situ hybridization for detection of all species of mRNA was performed at a final probe concen-
2.4. Electrophysiology Whole cell recordings were obtained at 348C in rat hippocampal slices as previously described w37x. Where ClC-2 activation was investigated, Naq, Kq, and Ca2q y conductances, HCOy exchange, and cation-Cly co3 –Cl transport were blocked by using artificial cerebrospinal
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fluid ŽACSF. composed of 109 mM NaCl, 1.25 mM NaH 2 PO4 , 10 mM glucose, 20 mM tetraethylammonium chloride, 2.5 mM CsCl, 5 mM 4-aminopyridine, 4 mM MgCl 2 , 26 mM N-Ž2-hydroxyethyl.piperazine-N X-Ž2ethanesulfonic acid. ŽHEPES., and 0.5 mM furosemide. The pH was adjusted to 7.4 with NaOH, and the ACSF was saturated with 100% O 2 . Electrode solutions included 120 mM CsCl, 2 mM MgCl 2 , 7 mM NaCl, 2 mM QX314, 10 mM HEPES, 1 mM potassium ethylene glycol-bisŽbaminoethyl ether. N,N,N X ,N X-tetraacetic acid ŽEGTA., 4 mM potassium adenosine 5X-triphosphate, and 0.3 mM sodium guanosine 5X-triphosphate; pH 7.2. When the resting membrane potential ŽRMP. was measured, ACSF was composed of 126 mM NaCl, 2.5 mM KCl, 26 mM HEPES, 2 mM CaCl 2 , 2 mM MgCl 2 , 1.25 mM NaH 2 PO4 , 10 mM glucose, and 0.5 mM furosemide; pH 7.4; Kq replaced Csq in the electrode solution, and QX314 was not used. Leak subtraction for ClC-2 exploited the slow activation and inward rectification of this conductance: the clamp current within 50 ms of stepping to the test voltage was subtracted from the steady-state current Žafter 4 s at the test potential.. Normalized, leak-subtracted currents were fit to a Boltzman equation as described previously w37x. Because GABA A conductances and known voltage-dependent Cly conductances do not inwardly rectify, they do not contribute to this estimation of the ClC-2 conductance.
3. Results During early brain development, intracellular Cly concentrations are maintained in neurons and neuronal precursors at levels that are much higher than the passive equilibrium concentration ŽE cl . predicted by the resting membrane potential and at this stage GABA A activation results in strong depolarization w18,24x. Inwardly directed cationCly cotransporters are suggested as the likely mechanism for Cly accumulation. With maturity, neuronal Cly levels change significantly such that GABA A activation results in a hyperpolarizing Cly influx. In earlier studies it was shown that de novo expression of ClC-2 in embryonic DRG neurons via viral vector counteracted the ability of the neuron to accumulate Cly thereby decreasing its concentration to that predicted by E cl w36x. These results suggest that ClC-2 expression may contribute to the development of a mature pattern of Cly homeostasis. To understand the contribution of ClC-2 expression to development of mature neuronal Cly homeostasis, both molecular detection of expression of ClC-2 and functional studies were performed. 3.1. ClC-2 mRNA expression is deÕelopmentally regulated in rat brain To determine the developmental time course of ClC-2 mRNA expression, ribonuclease protection analysis was
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Fig. 1. Ribonuclease protection analysis of ClC-2 expression in developing and adult rat brain. RPA analysis was performed by hybridizing 10 m g of total RNA with 32 P-labeled riboprobes which protected unique regions of ClC-2 ŽA. and the cellular housekeeping gene cyclophilin ŽB.. In lane 1 hybridization with total yeast RNA was performed as a control. Lanes 2–7 demonstrate results of hybridization with total RNA from E15, P0, P3, P7, P21, and adult rat brains. A. ClC-2 expression increases from a low at E15 to a peak at P3 which is maintained through adulthood. Note the lack of expression in yeast total RNA. B. Expression of the cellular housekeeping gene cyclophilin is not present in yeast and does not vary during development.
performed on total RNA obtained from rat brains ranging in age from E15 to adult. As shown in Fig. 1A, ClC-2 mRNA was detected at moderate levels in E15 brain Ž38% of adult., reaching peak levels by day P3, and was maintained at similar levels throughout postnatal development and into adulthood. Simultaneous hybridization of total brain RNA with probes for ClC-2 and cyclophilin ŽFig. 1B. detected unique protected fragments of expected sizes Žsee materials and methods.. Levels of cyclophilin, a cellular housekeeping gene, in the nuclease protection assay were used to confirm that equivalent amounts of RNA were used in each hybridization reaction. The 5X coding region probe used for ISHH described above was also used for RPA analysis Ž211 bp protected fragment. with identical results Ždata not shown.. These results indicate that significant levels of ClC-2 were expressed during the appearance of cortical neurons. 3.2. Localization of ClC-2 expression in deÕeloping brain The use of whole brain RNA to measure ClC-2 expression can obscure potentially important regional differences in ClC-2 expression. The level of ClC-2 expression varies greatly in neuronal populations in the adult brain w34x. In situ hybridization methods were used to examine the pattern of ClC-2 expression during development and to determine if significant differences exist in abundance of ClC-2 expression. 3.2.1. Cortex Embryonic brains of E15 rats demonstrated ClC-2 in situ hybridization signal in a small percentage of the
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Fig. 2. ClC-2 mRNA is expressed in neuronal precursors in prenatal rat brain. The distribution of ClC-2 mRNA was examined in embryonic day 15 and 18 rat brain using in situ hybridization methods. ŽA. E15 brain showing the cortical and striatal germinal matrices Ži.e. ventricular zone, VZ., surrounding the lateral ventricle. Scattered ClC-2 expressing cells can be seen within these regions in addition to limited signal within the beginnings of the cortical plate ŽCP.. ŽB. E18 brain demonstrates strongly labeled cells scattered throughout the ventricular zone. Diffuse staining was seen within the cortical plate just below the marginal zone ŽMZ, arrow. and a thin, lightly labeled subplate layer ŽSP, arrow. at the inner border of the CP. ŽC. Higher power view of E15 brain depicted in ‘A’ clearly showing scattered labeling in the VZ Žbracketed by arrows. as well as labeled cells in the newly formed CP Žarrowheads.. ŽD. Higher power view of E18 brain depicted in ‘B’ showing scattered labeling within the VZ with an accumulation of labeled cells demonstrated at its outer border Žarrowheads.. ŽE. E15 hippocampus showing ClC-2 label within the developing CA1 region Žarrow.. ŽF. ClC-2 expression in E18 hippocampus. The CA1 region Žarrow. is heavily labeled while dentate gyrus ŽDG. is relatively devoid of signal. Scale bar in A s 200 m m and applies to B, E, and F. Scale bar in C s 100 m m and also applies to D.
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Fig. 3. Expression of ClC-2 mRNA in cerebral cortex is developmentally regulated. ClC-2 mRNA expression was examined by in situ hybridization in coronal sections through cortex near the lateral ventricle from postnatal ages P0-adult. ŽA. P0: ClC-2 hybridization signal was significantly increased above embryonic levels. Heaviest labeling is seen within a narrow band within the CP Žasterisk., the supraventricular subplate region Žarrowhead., and a now very limited germinative VZ Žarrow.. ŽB. P0: Control hybridization performed with a sense-strand probe demonstrates absence of signal. ŽC. P7: All cortical layers are present. Heaviest labeling can be seen within a thin CP band at the border of the marginal zone Žasterisk., within layer V neurons, and in the subplate region Žarrowhead.. ŽD. P14: ClC-2 hybridization signal is evident in most cells with heaviest label in layer V and in the now limited subplate layer Žarrowhead.. ŽE. Adult: Most neurons contain some ClC-2 signal with the most prominent label still evident within layer V, however the subplate layer is no longer in evidence. Scale bars in all figuress 200 m m.
Fig. 4. Developmental changes in ClC-2 mRNA expression in caudoputamen ŽCPU.. Examination of ClC-2 hybridization within coronal sections through the CPU from ŽA. embryonic day 18 ŽE18., and ŽB. adult rat brains. These results demonstrate that the widespread embryonic expression of ClC-2 mRNA becomes markedly restricted to a limited population of neurons in the adult. The scale bar in A is 200 m m and also applies to panel B.
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densely packed cellular profiles of the proliferative neuroepithelium or ventricular zone ŽVZ. of developing cortex ŽFig. 2.. The majority of cells within this region did not demonstrate significant clc-2 staining consistent with the published observations of chloride accumulation and GABA A -mediated depolarization w18x. A similar pattern of clc-2 expression was observed in E18 brains within the VZ, although strongly positive profiles were apparent at the boundary of the ventricular and subventricular zones ŽFig. 2D.. Only rare ClC-2 positive profiles were detected in the subventricular zone, which largely gives rise to glial populations Žfor review see w10x.. The absence of ClC-2 expression in the subventricular zone is consistent with the lack of ClC-2 expression in glial regions in adult rat brain w34x. In contrast, significant levels of expression were present in the cortical plate and subplate neurons ŽFig. 2A, B, and C.. Similarly, ClC-2 hybridization signal was present in the region of the cortical plate that gives rise to the CA1 subfield but was not expressed to detectable levels in the secondary germinal cell mass that generates dentate
gyrus ŽFig. 2E and F.. These results suggest that ClC-2 expression is confined to cells committed to neuronal differentiation. From P0–P7 increasing signal was detected in virtually all cortical layers including remaining neurons of VIb ŽFig. 3A and C.. By P14, all cortical layers seen in the adult are distinct and express ClC-2 ŽFig. 3D.. However, a decline in the intensity of the ClC-2 signal in some layers was evident with the highest intensity of signal in layer V. ClC-2 signal levels declined further with maturity ŽFig. 3E. however, expression remained high within layer V as previously reported w34x. Restriction of cortical ClC-2 to larger output neurons is a theme repeated in other areas however, the timing and extent of change in expression was different in different regions of brain. 3.2.2. Caudoputamen The pattern of expression of ClC-2 observed in developing cortex suggested that expression of ClC-2 during development was related to the timing of neuronal differenti-
Fig. 5. In olfactory bulb, ClC-2 expression becomes restricted primarily to mitral cells during postnatal development. ClC-2 hybridization signal within coronal andror sagittal sections through the rat olfactory bulb ŽOB.. At postnatal day 0 ŽP0, A and D. proliferating andror migrating cells expressed ClC-2 mRNA throughout the OB with the exception of the olfactory glomeruli ŽGL.. Heaviest labeling is seen within the very broad mitral cell layer ŽMCL.. By postnatal day 7 ŽP7, B and E. labeling is much more restricted. However, the mitral cell layer is still thickened with prominent labeling just under and surrounding the glomeruli Žasterisk.. The unlabeled olfactory nerve layer ŽONL. can be seen just external to the glomeruli. In the adult ŽC and F. ClC-2 labeling is restricted to a 1 or 2 cell thick mitral cell layer with some labeling of cells seen just internal to the glomeruli. Scale bars in A, B, and C s 200 m m, D, E, and F s 100 m m.
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ation. The differentiating striatal and pallidal regions were easily recognizable at E18 ŽFig. 4A. with most of the cells within these structures demonstrating moderate to high levels of ClC-2 hybridization signal. Expression of ClC-2 was significantly reduced in most neurons of striatum by early postnatal life, although a limited population retained expression until adulthood ŽFig. 4B.. These data provide clear evidence of selective down-regulation of expression as the major mechanism for establishing postnatal and adult patterns of ClC-2 expression w34x. 3.2.3. Olfactory bulb In structures with significant postnatal neurogenesis and migration, such as olfactory bulb and cerebellum, ClC-2 expression was delayed. ClC-2 expression in the olfactory
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bulb was first observed on embryonic day 18 and restricted to the developing mitral cell layer Ždata not shown. whose resident neurons are the earliest born w1x. By P0 however, cells within all areas of the bulb exhibited heavy labeling with the exception of the glomeruli ŽFig. 5A and D.. The most intense signal was associated with the mitral cell layer with numerous smaller, well-labeled cells in evidence external to and internal to this layer. Between P7 and P14 ŽFig. 5B and E. the pattern of ClC-2 expression became more restricted, primarily limited to mitral cells in the adult ŽFig. 5C and F.. 3.2.4. Cerebellum By E18 ClC-2 hybridization signal was first detected in the neurons of the deep nuclei of cerebellum Ždata not
Fig. 6. Developmental changes in ClC-2 expression within the cerebellum. In the postnatally developing cerebellum ClC-2 hybridization signal occurred within the majority of cells but with maturity became restricted to Purkinje neurons. At postnatal day 3 ŽP3, A and C. heaviest label can be seen within the subpial external germinal layer ŽEGL., the multi-cell thick, prenatally formed Purkinje cell layer underneath ŽP., and cells within the deep nuclei Žasterisk.. The unlabeled band between the EGL and the Purkinje cell layer is the beginning of the molecular layer ŽM.. Post migratory granule cells ŽG. lie underneath the Purkinje cell layer and can be seen to be labeled as well. Other heavily labeled cells Žlikely in transit. can also be identified. In the adult ŽB and D., ClC-2 mRNA becomes restricted to the monolayer of Purkinje cells and what appears to be some externally displaced smaller cells within the molecular layer. Scale bars in A and B s 200 m m and scale bars in C and D s 100 m m.
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shown.. These neurons remained heavily labeled in the adult animal. Between P3 and P7 cells within the external germinal layer and Purkinje cell layer were heavily labeled ŽFig. 6A and C.. By P21 the adult pattern of ClC-2 labeling developed with the most intense signal in Purkinje cells, with some occasional labeling detected in large cells within the granular layer and a few scattered cells in the molecular layer ŽFig. 6B and D.. This pattern remained in all subsequent ages examined. 3.2.5. Hippocampus From embryonic ŽFig. 2E and F. to adult animals ŽFig. 7. pyramidal neurons of Ammon’s Horn Žfields CA1-4. demonstrated marked ClC-2 signal. In dentate gyrus, ClC-2 was not expressed prenatally nor to any significant degree in the adult. However, a thin layer of cells in the region of the earliest maturing neurons of the dentate gyrus transiently expressed ClC-2 hybridization signal during the first weeks of postnatal development ŽFig. 7C.. By P14 the
expression of ClC-2 was restricted to Ammon’s Horn and had assumed an adult-like distribution as described in previous work w34x ŽFig. 7B, D, and F.. 3.3. Functional expression of ClC-2 In previous studies we have correlated ClC-2 expression with the presence of a characteristic inwardly rectifying conductance in hippocampus of brain slices prepared from adult brains w35,37x. The presence of high levels of ClC-2 in pyramidal neurons of hippocampus during the first week of postnatal life would not be predicted if the GABAmediated depolarization that has been reported for CA3 w4,5,7x is due to intraneuronal chloride accumulation. An inwardly rectifying chloride conductance was present in P3–4 CA1 pyramidal cells ŽFig. 8A. consistent with the local expression of ClC-2 described above. This conductance was smaller than in adult pyramidal cells, but still large enough to prevent substantial chloride accumulation
Fig. 7. Developmental changes in ClC-2 mRNA expression in hippocampus. As determined by in situ hybridization, ClC-2 was expressed within most neuronal populations of the hippocampus by P0. However, expression was restricted to cells within Ammon’s Horn with maturity. In panel A the extent of ClC-2 expression can be seen in the P0 hippocampus. Heavy labeling can be identified within Ammon’s Horn ŽCA1 and CA3., the hilar region ŽH. and dentate gyrus ŽDG, arrows.. In panel C Žmagnified view of panel A. the extent of labeling is more apparent and migrating granule cells can be seen forming a line underneath CA1 Žarrow.. Positively labeled cell can also be identified along the hippocampal fissure Žasterisk.. Panel B Žadult, 9 months. demonstrates the extent to which ClC-2 expression becomes limited with maturity. Panel D Žmagnified view of panel B. shows the loss of ClC-2 expression within dentate granule cells. Note that expression of ClC-2 within choroid epithelia is evident by P0, and is maintained into adulthood Žasterisk in panels A and B.. The scale bars in A and B s 200 m m; the scale bars in C and D s 100 m m.
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Fig. 8. Electrophysiological analysis of ClC-2-mediated currents in developing rat brain. ŽA. The maximal conductance calculated for an inwardly rectifying chloride current in CA1 pyramidal cells from P3 Ž n s 8. and adult Ž n s 5. rats. The ClC-2 conductance was calculated as previously described w36,37x. ŽB. The ClC-2 conductance was large enough to prevent chloride accumulation in P3 neurons. The resting membrane potential of a P3 pyramidal cell was gradually depolarized due to chloride efflux via ClC-2 when recorded with a 135 mM chloride electrode solution Ž n s 2; w37x.. In contrast, the RMP of P3 pyramidal cells increased or was unchanged in 6 recordings with electrode solutions containing 4 mM chloride.
in pyramidal cells recorded with high-chloride electrodes ŽFig. 8B..
4. Discussion In this study, RPA analysis demonstrated a 2–3 fold increase in ClC-2 mRNA levels between E15 and early postnatal life. The levels of mRNA measured in total brain RNA were likely to under-represent the increases in neurons, since glial components contribute increasingly to brain mass later in development. In most regions of brain examined ClC-2 expression was initially diffuse, but later became restricted to specific neuronal populations. The in situ hybridization methods may not allow direct comparison of expression at different developmental stages; however, striking differential expression emerged as brain structures matured. In late developing structures, such as cerebellum and olfactory bulb, expression of ClC-2 was delayed. Large projection neurons, including mitral cells of the olfactory bulb, Purkinje cells of the cerebellum, pyramidal neurons of cortex and hippocampus, were the primary neurons within their respective structures that main-
tained high levels of ClC-2 expression. Virtually all neurons demonstrated expression early in development, although down-regulation of mRNA levels was evident in striatum by early postnatal life. The timing and localization of increasing ClC-2 expression was consistent with the transition from high intracellular Cly to levels approaching a passive distribution that has been reported during the transition from late embryonic to early postnatal life in cerebral cortex w24x. The ventricular zone of developing cortex gives rise to cells committed to differentiate into cortical neurons. GABA A agonists applied to cell populations from ventricular zone depolarize them to a degree sufficient to activate voltage-sensitive Ca2q channels w18,23x. Consistent with this observation, the majority of cell profiles in this region were negative for ClC-2 expression ŽFig. 2., since expression of ClC-2 would be expected to prevent Cly accumulation which is necessary for GABA A -mediated depolarization. The significance of the observed expression of ClC-2 in a subpopulation of ventricular zone cells is unknown. However, this could provide a mechanism for altering the response to GABA signaling in selected cortical precursors thereby preventing the GABA induced depolarization nor-
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mally observed in this region. Neurotransmitter induced activity can alter the migratory behavior of cortical precursors w2,3,14,43x as well as affect DNA synthesis w18x likely via Ca2q-mediated mechanisms. In other regions of the brain, such as the cerebellum and olfactory bulb, migrating populations of neuronal precursors also demonstrate ClC-2 signal ŽFig. 6A and D.. In contrast, regions that primarily give rise to glia such as the cortical subventricular zone did not demonstrate hybridization signal with the exception of rare profiles which may represent neuronal precursors in transit to cortical plate or the small percentage subventricular precursor cells that become neurons Žsee w11x for review.. Throughout development, high level expression of ClC-2 was confined to neuronal regions, although it remains possible that much lower levels of expression may occur in some glial populations that are below the limit of detection of in situ techniques. The ClC-2 mRNA expression observed during the first postnatal week of life was correlated with presence of a functional conductance with the same properties as observed following viral-mediated gene transfer to non-ClC-2 expressing neurons and observed in adult hippocampal pyramidal cells w35–37x. In pyramidal neurons of hippocampus, GABA A -mediated depolarization continues after the appearance of significant levels of ClC-2 w4,5,7x. Several possible mechanisms could account for this apparent contradiction and contribute to GABA A -mediated depolarization after ClC-2 expression. First, ClC-2 is subject to regulation by stimuli that include changes in extracellular pH, exogenous Zn2q, and activators of protein kinase C w36,37x, and may not be active under the conditions utilized for these studies. Secondly, flux of bicarbonate via the GABA A -gated anion channel can produce a significant depolarization after collapse of Cly gradients w38x. Regardless of the specific explanation for the GABA A -mediated depolarization, the experimental evidence clearly confirms the presence of ClC-2, which should oppose neuronal accumulation of Cly. The functional role of ClC-2 may vary significantly throughout development. In addition to playing a role in the prevention of Cly accumulation, this channel has also been proposed to serve as a volume-regulator in non-neuronal tissues. The characterization of ClC-2 expressed in Xenopus oocytes demonstrates activation in response to extracellular hypotonic solutions w9x. Activation of ClC-2 and swelling activated Kq channels by hypotonic stimuli provides a potential mechanism for regulatory volume decrease ŽRVD. mediated by the outward movement of Cly and Kq w9,26,28,30,40x. In adult neurons, ClC-2 activation by intracellular Cly concentration is a significant feature of regulation w34,36,37x. At early stages of development, ClC-2 may have a more significant role in mediating volume-regulation in neuronal precursors than in the mature neurons, where control of intracellular Cly concentration is a significant effect. Functional studies of ClC-2 activation demonstrated a smaller absolute conduc-
tance activated by high intracellular Cly in P3 hippocampus; however, even at this stage of development ClC-2 was sufficient to strongly stabilize intracellular Cly concentration near E Cl . Control of ClC-2 by separate regulatory mechanisms may allow expression of ClC-2 in a sub-population of neuronal precursors that maintain high intracellular Cly w18,24x. In adult brain transmembrane neuronal Cly gradients are maintained at values that are near or below the resting membrane potential as a necessary condition for GABA A receptor-mediated postsynaptic inhibition. This transmembrane chloride gradient is not favorable for regulatory volume decrease mediated by ClC-2 activation and subsequent chloride efflux. An alternative function for ClC-2 is suggested by the varied experimental response to chloride loading by neurons that differentially express this channel w34,37x. In adult hippocampus the dentate gyrus granular neurons, which lack ClC-2, can be readily loaded with Cly in whole cell clamp studies, while pyramidal neurons resist loading with Cly because of the activation of ClC-2 w37x. In neurons that express ClC-2, activation by increasing intracellular Cly has been demonstrated under isotonic conditions w37x. In embryonic DRG neurons, the de novo expression of ClC-2 by virus vector-mediated gene transfer resulted in a significant shift in neuronal Cly concentrations prior to any change in cell volume w36x. Neurons have special requirements to regulate intracellular Cly separate from cell volume, since the transmembrane Cly concentration gradient is a primary determinant of GABA A -mediated signal transduction. The differential expression of ClC-2 in neurons during maturation is a striking feature that has not been reported for other voltage-gated-chloride channels w12x. These findings indicate that components of Cly homeostatic control change over a significant period of life and may contribute to homeostatic changes in adult or aging brain. These observations suggest that certain classes of neurons have retained ClC-2 as a high capacity system to prevent Cly loading from occurring. The mechanisms that control ClC-2 expression are unknown; however, ClC-2 expression is precisely regulated in tissues such as lung with marked shut-down in expression within 24 h of birth w22x. In contrast, the changes in ClC-2 expression observed in rat brain extend over a long time period and involve both cell-specific increases and decreases in expression during development. The net effect is that the total abundance of ClC-2 expression is little changed from early postnatal to adult life in rat brain. The ontogeny of neuronal Cly homeostasis involves not only anion channels but also involves cation-Cly co-transport and anion-exchange mechanisms. The cation y Cly co-transporters, which couple movement of Cly to cationic gradients, are likely to be significant both early in development and during post-natal life w18,24x. The initially depolarizing actions of GABA A agonists on neuronal precursors are dependent on active accumulation of Cly against
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the concentration gradient by a furosemide-inhibitable mechanism suggesting a role for inwardly directed cationCly cotransport w18,24x. This process likely requires unique inwardly directed cation-Cly cotransporters. In previous studies we have demonstrated that de novo expression of ClC-2 using a viral vector is sufficient to stabilize neuronal Cly concentrations near E Cl in cultured embryonic DRG neurons which normally actively accumulate Cly and depolarize in response to GABA A activation w36x. During the period when increased ClC-2 expression was observed, Cly concentrations in neurons approach concentrations predicted by a passive distribution, suggesting a role for ClC-2 during this stage of development. Postnatally, the intraneuronal concentration of Cly decreases until an adult pattern is attained with Cly maintained at concentrations less than predicted by E Cl w24x suggesting a role for outwardly directed transport mechanisms such as the recently described Kq dependent cation-Cly cotransporters, KCC-1 and KCC-2 w8,27x. Anion exchange mechanisms may also significantly contribute to the developmental changes in Cly homeostasis within the nervous system. These proteins have been identified in neuronal and nonneuronal cells, have been shown to be developmentally regulated in some populations, and have been suggested to play a role in neural induction w13,15,17,29,33x. Therefore, changes in ClC-2 expression are likely to be one component that contributes to developmental alterations in neuronal chloride homeostasis and the reversal of GABA A mediated depolarization.
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Acknowledgements This work is supported by grants from the NIH including a training grant NS 0732 to GHC, NS 34360 to KJS, and a grant from the March of Dimes 1-FY- 0454 to RLS.
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