Neuron,
Vol. 7, 927-936, December,
1991, Copyright
0 1991 by Cell Press
Expression Patterns of GABA* Receptor Subtypes in Developing Hippocampal Neurons Iris Killisch,* Carlos C. Dotti,+ David J. Laurie,* Hartmut Liiddens,* and Peter H. Seeburg* *Laboratory of Molecular Neuroendocrinology Center for Molecular Biology University of Heidelberg Im Neuenheimer Feld 282 +EMBL Meyerhofstrasse 1 6900 Heidelberg Germany
Summary Developing cultured hippocampal neurons were studied for the expression of CABA* receptor subunits using anti-peptide antibodies. The antibodies decorated the plasma membrane of cell bodies and all neurites in immature neurons, whereas the immunoreactivity was restricted to somata and dendrites in fully developed cells. Most receptor subunits were expressed throughout the entire culture period. However, the al and y2 subunits were detected only late in culture, while a6 disappeared around this time. These findings were partly corroborated by in situ hybridization data in the developing rat hippocampus. Our results reveal a temporal control of gene expression for subsets of receptor subunits in hippocampal neurons and suggest a change in the subunit composition of CABAA receptors in the developing brain. Introduction In the mammalian brain, fast inhibitory neurotransmission is largely mediated bythey-aminobutyricacid type A receptor (GABA*R). This multisubunit complex contains a Cl- ion-conducting channel whose transmitter-gated activity can be modulated by compounds used clinically in treating epilepsy, anxiety, and insomnia. Foremost among such compounds are benzodiazepines, which bind with high affinity to the GABAAR (reviewed in Olsen and Venter, 1986). Recent cloning studies have elucidated a family of sequence-related receptor subunits (a, B, y, and 6) and their respective variants (al to a6; Bl to (33; ~1 and ~2) (reviewed in Olsen and Tobin, 1990; Luddens et al., 1990). Each mRNAshows marked anatomical specificities in its distribution (Wisden et al., 1988; Shivers et al., 1989; Khrestchatisky et al., 1989), suggesting shifting partnerships in receptors across different brain areas. Taken together these findings predict a considerable diversity of GABAxRs (Seeburg et al., 1990). Indeed, recombinant expression studies have demonstrated that the functional properties of GABAARs depend on subunit composition. Receptors assembled from members of three subunit classes (a, B, and y) display large single-channel conductances (Ver-
doorn et al., 1990) and benzodiazepine pharmacologies differing with the particular variants present (Pritchett et al., 1989a; Pritchett and Seeburg, 1990; Luddens et al., 1990). Receptors composed only of a and B subunits fail to respond to benzodiazepines and have smaller unitary conductances (Pritchett et al., 198913; Verdoorn et al., 1990). In analogy to the recognized subunit switch in the nicotinic acetylcholine receptor of developing muscle (Mishina et al., 1986), temporal changes in GABAAR subunit expression and hence in receptor composition maybecrucialforestablishinginhibitorycircuitry in the maturing brain. Little is known regarding the developmental expression of these subunits, although differences in Zn2+ sensitivity (Westbrook and Mayer, 1987), benzodiazepine pharmacology (Sato and Neale, 1989), and patterns of photoaffinity-labeled subunits (Eichinger and Sieghart, 1986; Vitorica et al., 1990) reported for embryonic and adult GABAARs indicatedistinct changes during brain development. In an attempt to trace temporal expression patterns of GABAAR subunits in a culture system, we have analyzed embryonic hippocampal neurons using as probes a set of subunit-specific antisera. These neurons, like their in situ counterparts, develop in a distinct morphological progression from nonpolarized to fully mature cells. Early on, the cells extend lamellipodia (stage I) and form short processes (stage 2). Within 24 hr, the neurons polarize by elongating one of their minor processes, which differentiates into the axon (stage 3). After 5-10 days, the remaining neurites become dendrites (stage 4), and by 14 days, the fully matured neurons (stage 5) form extensive synaptic connections (Banker and Waxman, 1988; Dotti et al., 1988). We now report that, with progressing maturation, these neurons switch on, or switch off, the expression of different GABAAR subunits while other subunits of this family remain unchanged in their expression.Adevelopmental regulation of GABAAR subunit expression was also revealed by in situ hybridization in the rat hippocampus. A comparison of culture and in situ results showed important subunit-specific correlations in the developmental program of hippocampal neurons. Results Antisera for CABAAR Subunits Antisera were raised against a series of synthetic peptides coupled to keyhole limpet hemocyanin via an additional N-terminal cysteine residue. The peptide sequences specific for the al (Schofield et al., 1987), a3 (Levitan et al., 1988), a5 (Khrestchatisky et al., 1989; Pritchett and Seeburg, 1990), and a6 (Luddens et al., 1990) variants as well as for the B2 (Ymer et al., 1989), y2 (Pritchett et al., 1989b; Shivers et al., 1989), and 8 (Shivers et al., 1989) subunits corresponded either to
Neuron 928
the predicted mature N-termini ((32, 72, and 8) or to C-terminal sequences (a variants) (Table 1). The N-terminal sequence of the 82 subunit is very similar to that of the Bl and B3 variants (Ymer et al., 1989), and hence the respective antiserum may cross-react with these B subunits. All peptide epitopes are extracellularly located according to the accepted topology for GABAAR subunits (Unwin et al., 1990). The use of the al- and a6-specific antisera has been reported previously (Liiddens et al., 1990, 1991), and similar subunit-specific anti-peptide antisera were constructed also by others (Duggan and Stephenson, 1989; Fuchs et al., 1990). All anti-peptide antibodies were tested in immunoblotsof cell membranes from primary hippocampal neurons resolved by SDS-PAGE (Figure 1). Each antibody reacted with single polypeptides with M, values of 51,000 (al), 59,000 (a3), 58,000 (a5), 56,000 (a6), 57,000 (B), 53,000 (y2), and 52,000 (8). These proteins were not detected upon coincubating the antiserum with the peptides used as antigens (Figure 1). The polypeptide sizes visualized by these antisera were compatible with those predicted bythe respective cloned cDNAs. The antisera detected proteins of the same sizes in immunoblotted membranes from ratadult hippocampus and cerebellum and recognized the receptors in their native conformation, as demonstrated by their ability to immunoprecipitate [3H]muscimol binding sites from brain (data not shown).
Table 1. Peptide Antigens Specific for Different GABAAR Subunits Corresponding to Predicted Mature N-Terminal (f32, ~2, and S Subunits) or C-Terminal (a Subunits) Sequences
3 + -
+
4 -
+-
5 +-
EPQLKAPTPHQ ESAIKGMIRKQ NREPVIKGATSPK SKDTMEVSNSVE QSVNDPSNMSLVKET QKSDDDYEDYAS NDICDYVCSNLEISW
;; 8
-
Sequence
Antibodies Cl4 c3 c5 C6 NB NIO Nil
neurons (data not shown) and may retlect the coexistence of pyramidal cells from different hippocampal regions (Rothman and Cowan, 1981). In immature hippocampal neurons of stage 2, subunit-specific immunoreactivity was observed intracellularly in cell bodies and all neurites (Figure 2A). At stage 3, when axons were extended, the antisera decorated cell bodies and short neurites, including the elongated axons (Figure 26). These results indicate that, up to this developmental stage, polarized sorting of the GABAAR subunits had not occurred. However, when axons and dendrites werefullydeveloped (stage 5,15 days after plating), the axonal staining had disappeared while somata and dendrites reacted strongly with the antisera (Figures 2C and 2D). This polarized distribution of the receptor was first observed in neurons after 7 days in culture. As GABAARs form functional channels only when inserted into the plasma membrane, we analyzed the presence of subunit epitopes on the cell surface. Using the S-specific antibody, surface labeling of immature neurons of stage 3 (3 days after plating) resulted in a punctate labeling spread over the entire cell, indicating that the receptor is inserted in the plasma membrane (Figure 2E). Similarly, the antibodies for the a3,
6 +-
Peptide
al a3 a5
Y2
Cellular Localization of GABAAR Subunits Hippocampal cells were plated on poly-L-lysinecoated coverslips and analyzed by indirect immunofluorescence for the cellular distribution of the GABAAR subunits. In these studies, not all cells showed positive immunoreactivity for all subunits. This was most noticeable for the a3-specific antiserum, which labeled approximately one-third of the
12 + -
Subunits
7 +-
Figure 1. lmmunoblot with Rabbit AntiPeptide Antisera Specific for the GABAAR Subunits Proteins of hippocampal primary cells wereelectrophoresed, and gelswere transferred to PVDF membranes and probed with immune sera, as described in Expertmental Procedures. Lanes 1-7were probed with the antisera in the following order: Cl4 (al), C3 (a3), C5 (a5), C6 (a6), N8 (f32), NIO fy2), and Nil (S), either preadsorbed with the corresponding peptide antigen at lOug/ml(-)orin theabsenceoftheantigen f+). Molecular weight standards (in kilodaltons) are indicated on the left,
GABA* Receptors 929
in Development
Figure 2. CABAAR B Subunit
Expression
in Cultured
Hippocampal
Neurons
(Stages 2-5)
(A-D) Cells were fixed in paraformaldehyde, permeabilized with Triton X-100, and stained with N8 antiserum. At stage 2, the cell body and all minor processes (arrowheads) are labeled (A). At stage 3, the antiserum stains short dendrites (arrowheads) and the developing axon (arrow) (B). In differentiated cells (stage 5), the labeling is restricted to the cell body and dendrites (D). Dendrites (arrowheads) and axons (arrows) are distinguished by phase-contrast microscopy (C). Cells were incubated with NB antiserum for 30 min at 37V, fixed with paraformaldehyde, and processed for immunofluorescence. Immature neurons (stage 3) show a punctate labeling on cell body, dendrites (arrowheads), and the long axon (arrow), indicating the presence of B-containing CABA*Rs in the plasma membrane (E). In differentiated cells (stage 5) a nonuniform clustering of the reaction products was observed exclusively on cell bodies and dendrites (arrowheads) (F). Bars, 10 urn.
Neuron 930
a5, a6, and 6 subunits decorated young neurons (
Vol. 7, 927-936, December,
1991, Copyright
0 1991 by Cell Press
Expression Patterns of GABA* Receptor Subtypes in Developing Hippocampal Neurons Iris Killisch,* Carlos C. Dotti,+ David J. Laurie,* Hartmut Liiddens,* and Peter H. Seeburg* *Laboratory of Molecular Neuroendocrinology Center for Molecular Biology University of Heidelberg Im Neuenheimer Feld 282 +EMBL Meyerhofstrasse 1 6900 Heidelberg Germany
Summary Developing cultured hippocampal neurons were studied for the expression of CABA* receptor subunits using anti-peptide antibodies. The antibodies decorated the plasma membrane of cell bodies and all neurites in immature neurons, whereas the immunoreactivity was restricted to somata and dendrites in fully developed cells. Most receptor subunits were expressed throughout the entire culture period. However, the al and y2 subunits were detected only late in culture, while a6 disappeared around this time. These findings were partly corroborated by in situ hybridization data in the developing rat hippocampus. Our results reveal a temporal control of gene expression for subsets of receptor subunits in hippocampal neurons and suggest a change in the subunit composition of CABAA receptors in the developing brain. Introduction In the mammalian brain, fast inhibitory neurotransmission is largely mediated bythey-aminobutyricacid type A receptor (GABA*R). This multisubunit complex contains a Cl- ion-conducting channel whose transmitter-gated activity can be modulated by compounds used clinically in treating epilepsy, anxiety, and insomnia. Foremost among such compounds are benzodiazepines, which bind with high affinity to the GABAAR (reviewed in Olsen and Venter, 1986). Recent cloning studies have elucidated a family of sequence-related receptor subunits (a, B, y, and 6) and their respective variants (al to a6; Bl to (33; ~1 and ~2) (reviewed in Olsen and Tobin, 1990; Luddens et al., 1990). Each mRNAshows marked anatomical specificities in its distribution (Wisden et al., 1988; Shivers et al., 1989; Khrestchatisky et al., 1989), suggesting shifting partnerships in receptors across different brain areas. Taken together these findings predict a considerable diversity of GABAxRs (Seeburg et al., 1990). Indeed, recombinant expression studies have demonstrated that the functional properties of GABAARs depend on subunit composition. Receptors assembled from members of three subunit classes (a, B, and y) display large single-channel conductances (Ver-
doorn et al., 1990) and benzodiazepine pharmacologies differing with the particular variants present (Pritchett et al., 1989a; Pritchett and Seeburg, 1990; Luddens et al., 1990). Receptors composed only of a and B subunits fail to respond to benzodiazepines and have smaller unitary conductances (Pritchett et al., 198913; Verdoorn et al., 1990). In analogy to the recognized subunit switch in the nicotinic acetylcholine receptor of developing muscle (Mishina et al., 1986), temporal changes in GABAAR subunit expression and hence in receptor composition maybecrucialforestablishinginhibitorycircuitry in the maturing brain. Little is known regarding the developmental expression of these subunits, although differences in Zn2+ sensitivity (Westbrook and Mayer, 1987), benzodiazepine pharmacology (Sato and Neale, 1989), and patterns of photoaffinity-labeled subunits (Eichinger and Sieghart, 1986; Vitorica et al., 1990) reported for embryonic and adult GABAARs indicatedistinct changes during brain development. In an attempt to trace temporal expression patterns of GABAAR subunits in a culture system, we have analyzed embryonic hippocampal neurons using as probes a set of subunit-specific antisera. These neurons, like their in situ counterparts, develop in a distinct morphological progression from nonpolarized to fully mature cells. Early on, the cells extend lamellipodia (stage I) and form short processes (stage 2). Within 24 hr, the neurons polarize by elongating one of their minor processes, which differentiates into the axon (stage 3). After 5-10 days, the remaining neurites become dendrites (stage 4), and by 14 days, the fully matured neurons (stage 5) form extensive synaptic connections (Banker and Waxman, 1988; Dotti et al., 1988). We now report that, with progressing maturation, these neurons switch on, or switch off, the expression of different GABAAR subunits while other subunits of this family remain unchanged in their expression.Adevelopmental regulation of GABAAR subunit expression was also revealed by in situ hybridization in the rat hippocampus. A comparison of culture and in situ results showed important subunit-specific correlations in the developmental program of hippocampal neurons. Results Antisera for CABAAR Subunits Antisera were raised against a series of synthetic peptides coupled to keyhole limpet hemocyanin via an additional N-terminal cysteine residue. The peptide sequences specific for the al (Schofield et al., 1987), a3 (Levitan et al., 1988), a5 (Khrestchatisky et al., 1989; Pritchett and Seeburg, 1990), and a6 (Luddens et al., 1990) variants as well as for the B2 (Ymer et al., 1989), y2 (Pritchett et al., 1989b; Shivers et al., 1989), and 8 (Shivers et al., 1989) subunits corresponded either to
Neuron 928
the predicted mature N-termini ((32, 72, and 8) or to C-terminal sequences (a variants) (Table 1). The N-terminal sequence of the 82 subunit is very similar to that of the Bl and B3 variants (Ymer et al., 1989), and hence the respective antiserum may cross-react with these B subunits. All peptide epitopes are extracellularly located according to the accepted topology for GABAAR subunits (Unwin et al., 1990). The use of the al- and a6-specific antisera has been reported previously (Liiddens et al., 1990, 1991), and similar subunit-specific anti-peptide antisera were constructed also by others (Duggan and Stephenson, 1989; Fuchs et al., 1990). All anti-peptide antibodies were tested in immunoblotsof cell membranes from primary hippocampal neurons resolved by SDS-PAGE (Figure 1). Each antibody reacted with single polypeptides with M, values of 51,000 (al), 59,000 (a3), 58,000 (a5), 56,000 (a6), 57,000 (B), 53,000 (y2), and 52,000 (8). These proteins were not detected upon coincubating the antiserum with the peptides used as antigens (Figure 1). The polypeptide sizes visualized by these antisera were compatible with those predicted bythe respective cloned cDNAs. The antisera detected proteins of the same sizes in immunoblotted membranes from ratadult hippocampus and cerebellum and recognized the receptors in their native conformation, as demonstrated by their ability to immunoprecipitate [3H]muscimol binding sites from brain (data not shown).
Table 1. Peptide Antigens Specific for Different GABAAR Subunits Corresponding to Predicted Mature N-Terminal (f32, ~2, and S Subunits) or C-Terminal (a Subunits) Sequences
3 + -
+
4 -
+-
5 +-
EPQLKAPTPHQ ESAIKGMIRKQ NREPVIKGATSPK SKDTMEVSNSVE QSVNDPSNMSLVKET QKSDDDYEDYAS NDICDYVCSNLEISW
;; 8
-
Sequence
Antibodies Cl4 c3 c5 C6 NB NIO Nil
neurons (data not shown) and may retlect the coexistence of pyramidal cells from different hippocampal regions (Rothman and Cowan, 1981). In immature hippocampal neurons of stage 2, subunit-specific immunoreactivity was observed intracellularly in cell bodies and all neurites (Figure 2A). At stage 3, when axons were extended, the antisera decorated cell bodies and short neurites, including the elongated axons (Figure 26). These results indicate that, up to this developmental stage, polarized sorting of the GABAAR subunits had not occurred. However, when axons and dendrites werefullydeveloped (stage 5,15 days after plating), the axonal staining had disappeared while somata and dendrites reacted strongly with the antisera (Figures 2C and 2D). This polarized distribution of the receptor was first observed in neurons after 7 days in culture. As GABAARs form functional channels only when inserted into the plasma membrane, we analyzed the presence of subunit epitopes on the cell surface. Using the S-specific antibody, surface labeling of immature neurons of stage 3 (3 days after plating) resulted in a punctate labeling spread over the entire cell, indicating that the receptor is inserted in the plasma membrane (Figure 2E). Similarly, the antibodies for the a3,
6 +-
Peptide
al a3 a5
Y2
Cellular Localization of GABAAR Subunits Hippocampal cells were plated on poly-L-lysinecoated coverslips and analyzed by indirect immunofluorescence for the cellular distribution of the GABAAR subunits. In these studies, not all cells showed positive immunoreactivity for all subunits. This was most noticeable for the a3-specific antiserum, which labeled approximately one-third of the
12 + -
Subunits
7 +-
Figure 1. lmmunoblot with Rabbit AntiPeptide Antisera Specific for the GABAAR Subunits Proteins of hippocampal primary cells wereelectrophoresed, and gelswere transferred to PVDF membranes and probed with immune sera, as described in Expertmental Procedures. Lanes 1-7were probed with the antisera in the following order: Cl4 (al), C3 (a3), C5 (a5), C6 (a6), N8 (f32), NIO fy2), and Nil (S), either preadsorbed with the corresponding peptide antigen at lOug/ml(-)orin theabsenceoftheantigen f+). Molecular weight standards (in kilodaltons) are indicated on the left,
GABA* Receptors 929
in Development
Figure 2. CABAAR B Subunit
Expression
in Cultured
Hippocampal
Neurons
(Stages 2-5)
(A-D) Cells were fixed in paraformaldehyde, permeabilized with Triton X-100, and stained with N8 antiserum. At stage 2, the cell body and all minor processes (arrowheads) are labeled (A). At stage 3, the antiserum stains short dendrites (arrowheads) and the developing axon (arrow) (B). In differentiated cells (stage 5), the labeling is restricted to the cell body and dendrites (D). Dendrites (arrowheads) and axons (arrows) are distinguished by phase-contrast microscopy (C). Cells were incubated with NB antiserum for 30 min at 37V, fixed with paraformaldehyde, and processed for immunofluorescence. Immature neurons (stage 3) show a punctate labeling on cell body, dendrites (arrowheads), and the long axon (arrow), indicating the presence of B-containing CABA*Rs in the plasma membrane (E). In differentiated cells (stage 5) a nonuniform clustering of the reaction products was observed exclusively on cell bodies and dendrites (arrowheads) (F). Bars, 10 urn.
Neuron 930
a5, a6, and 6 subunits decorated young neurons (
