Developmental expression of the GIRK family of inward rectifying potassium channels: implications for abnormalities in the weaver mutant mouse

Brain Research 778 Ž1997. 251–264

Research report

Developmental expression of the GIRK family of inward rectifying potassium channels: implications for abnormalities in the weaÕer mutant mouse Shu-Cheng Chen

a,1

, Patricia Ehrhard b , Dan Goldowitz c , Richard J. Smeyne

d, )

a

d

Neurogenetics Program, Department of CNS Research, Hoffmann-LaRoche, 340 Kingsland Street, Nutley, NJ 07110, USA b Department of CNS Research, Hoffmann-La Roche, Basel, Switzerland c Department of Anatomy and Neurobiology, UniÕersity of Tennessee at Memphis, 875 Monroe AÕenue, Memphis, TN 38163, USA Department of DeÕelopmental Neurobiology, St. Jude Children’s Research Hospital, 332 North Lauderdale, Memphis, TN 38105, USA Accepted 15 July 1997

Abstract G-protein-gated inward rectifying potassium channels ŽGIRKs. are a newly identified gene family. These gene products are thought to form functional channels through the assembly of heteromeric subunits. Recently, it has been demonstrated that a point mutation in the GIRK 2 gene, one of the GIRK family members, is the cause of the neurological and reproductive defects observed in the weaÕer Ž wÕ . mutant mouse. The mechanismŽs. by which a single amino acid substitution in GIRK2 protein leads to the severe phenotypes in the wÕ r wÕ mouse is not fully understood. However, it implicates the importance of GIRK channels in neuronal development. To characterize the mRNA expression patterns of GIRK 1–3 during mouse brain development we have used in situ hybridization analyses. We found that the expression of all three genes showed developmental regulation. In most areas that showed expression, the levels of GIRK 1-3 transcripts reached their peak at around postnatal day 10 ŽP10.. In general, GIRK 1 showed the least fluctuation in its levels of expression during development, while dynamic changes were found with the levels of GIRK 2 and GIRK 3 transcripts. GIRK 3 becomes the predominant inward rectifying Kq-channel in the brain at later postnatal ages. In the CNS regions affected in the wÕ r wÕ mouse, GIRK 2 is the predominant inward rectifying channel that is expressed. This suggests that the presence of the other subtypes are able to compensate for the mutated GIRK 2 channel in weaÕer neurons that survive. q 1997 Elsevier Science B.V. Keywords: Weaver; Thalamus; Substantia nigra; Cerebellum; In situ hybridization; K IR channel

1. Introduction G-protein-gated inward rectifying Kq channels, GIRKs, are coupled to and regulated by neurotransmitter action and can play important roles in modulating neuronal excitability w1,9x. Molecular cloning has demonstrated that there are at least four different mammalian genes in this expanding family, GIRK 1–GIRK4 w2,11,15–18,28x. Structurally as well as physiologically, GIRK channels belong to a superfamily called the inwardly rectifying Kq chan-

)

Corresponding author. Fax: q1 Ž901. 495-3143; E-mail: [email protected] 1 Present address: Schering-Plough Research Institute, Immunology Department, 2015 Galloping Hill Road, Kenilworth, NJ 07033, USA. 0006-8993r97r$17.00 q 1997 Elsevier Science B.V. All rights reserved. PII S 0 0 0 6 - 8 9 9 3 Ž 9 7 . 0 0 8 9 6 - 2

nels ŽKir. which are distinct from the voltage-gated Kq channel superfamily w4x. This Kir family shares a conserved inner core structure ŽH5., responsible for ion conductance, with the voltage-gated channel family, but has only two membrane-spanning segments ŽM1 and M2.. Presently, the GIRK channels are thought to be heteromultimers consisting of 2–4 distinct GIRK gene products w12,15,18x. The first GIRK channel discovered, GIRK 1, was isolated from the rat atrium and brain by expression cloning. Using homology screening with the GIRK 1 probe, three more distinct mammalian GIRK genes, GIRK 2–4, have subsequently been cloned. Very recently, a new GIRK gene, XIR, cloned from a Xenopus oocyte cDNA library has been added to the GIRK family w8x. Northern blot analysis and in situ hybridization have shown that

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GIRK 1–3 messages are widely expressed in the adult brain w10,11x, while GIRK4 is predominantly expressed in the atrium of the heart w3x. Very recently, low levels of GIRK4 message expression was found in restricted areas of the adult brain w10x. Recently, the role of these channels in neuronal development has come under scrutiny with the revelation that a point mutation in the GIRK 2 gene is responsible for the neurological defects seen in the weaÕer Ž wÕ . mutant mouse w22x. The weaver phenotype is semi-dominant. The homozygous Ž wÕ r wÕ . mouse is characterized by severe ataxia, hyperactive behavior, tremor, reduced viability and male infertility. Anatomically, pronounced cell death in the nervous system of the wÕ mouse has been found in three cell types associated with motor function: the cerebellar granule and Purkinje cells and the dopaminergic cells of the substantia nigra w6,23,24,26,27x. In the testis, degeneration of germ and Sertoli cells has been reported in wÕ mice w7,34x. The heterozygous animal Ž wÕrq . has defects in the same areas, although they are attenuated compared to the wÕ r wÕ mouse. In the adult mouse, GIRK 2 is expressed in widespread populations of neurons w11,19x. However, it is not known how a mutation of one single nucleotide in the GIRK 2 gene can affect diverse populations of cells with no evident functional commonality. Results of recent studies on the pathophysiology of the weaÕer mutation regarding the action of the mutation found in the H5 domain of GIRK 2 are controversial. One school of thought suggests that the mutation results in a loss of its Kq-selectivity, leading to cell death via an influx of sodium into the cell w13,30x; while a second school of thought suggests that the activation of NMDA channels with coincident loss of Kq-inward rectifying current leads to a build-up of calcium in the cell which is directly related to its toxicity w29x. The former is supported by the observation that weaÕer granule cells could be saved through the use of pharmacological blockers of Naq influx w13x, while the latter hypothesis finds support in cell saving with NMDA antagonists and calcium blockers w31x. In developmental neurobiology, the role of ion channels in shaping the nervous system has remained an open question. However, changes in the density of voltage-gated ion channels during development has been documented w35x. It is possible that similar phenomena can be found for the G-protein-gated inwardly rectifying channels. In addition the identification of a mutated GIRK gene in the neurological weaÕer mouse implicates the activity of GIRK channels in neuronal development. Due to the multitude of GIRK genes and the possible assembly of these gene products to form heteromultimeric channels, it is important to document the cellular distribution of each GIRK message during development. In this report, we show the distribution patterns and expression levels of GIRK 1, GIRK 2 and GIRK 3 messages in the developing mouse brain from a late embryonic stage to 20 days after birth.

2. Materials and methods 2.1. Animals Inbred C57Blr6J mice were used in all experiments. Embryonic day 15.5 ŽE15.5. embryos were collected from timed-pregnant mice, placed on ice until deeply anesthetized and perfused transcardially with 4% paraformaldehyde in 1 = PBS ŽpH 7.4.. Newborn ŽP0. mice, sacrificed within 12 h of natural birth, were deeply anesthetized with Avertin and perfused as described above. All animal housing and experimental procedures were performed following the guidelines of Hoffmann-La Roche Institutional Animal Care and Use Committee. 2.2. PCR and riboprobes Antisense and sense transcripts of each riboprobe were generated by transcribing linearized plasmid DNA with T7 RNA polymerase ŽBoehringer Mannheim.. Plasmid constructs used for each riboprobe were generated by subcloning PCR fragments into the pNoTArT7TM vector Ž5 prime ™ 3 prime.. The PCR fragments of each GIRK cDNA were synthesized by reverse transcription-PCR ŽRT-PCR. using the total mRNAs isolated from adult mouse brain as templates. A 394-bp of GIRK 1 cDNA fragment was generated with the primers 5X-CCTTACAGCGTGAAAGAGCAGGAG-3X Žq1108 to q1131. and 5X-CTTCCATCCTGGTAGGTCCTCCAG-3X Žq1501 to q1478.. This PCR fragment was further digested with restriction enzymes to generate a 207 bp of PstI–HindIII fragment Žq1256 to q1462. and subcloned into pNoTArT7TM vector. This GIRK 1-pNoTArT7TM plasmid DNA was then used to generate the GIRK 1 riboprobe used in the in situ hybridization. The strength of the hybridization of this probe as measured by GC content was 42.99%. The stoichiometry of the probe by percent uracil content was 33.33%. The numbering of the GIRK 1 probe is based on the sequence reported in Genbank, accession code D45022. The GIRK 2-specific probe was amplified with the primers 5X-GAATGGAGTCTCCTGAAAGCCTGC-3X Žq285 to q308. and 5X-CTGATCGCCTCTCTCGGAACGGA-3X Žq439 to q417. resulting in a 155-bp fragment. The strength of the hybridization of this probe as measured by GC content was 50.31%. The stoichiometry of the probe by percent uracil content was 16.77%. The Genbank accession code for the GIRK 2 sequence is U11859. A 222-bp RT-PCR fragment of GIRK 3 cDNA was amplified with the primers 5XGCTGCGAGTGGAGGTGGAAGAAGAG-3X Žq1543 to q1567. and 5X-CCCCATGAGCCCATCCTTCTGTCC-3X Žq1764 to q1741.. The strength of the hybridization of this probe as measured by GC content was 56.48%. The stoichiometry of the probe by percent uracil content was 20.00%. The Genbank accession code for GIRK 3 is U11860. All of the probes were used at 1 = 107 c.p.m.rml.

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2.3. In situ hybridization Following perfusion, the mouse tissues were dissected and immersed in the same fixative overnight at 48C followed by submersion in 30% sucroser1= PBS. Ten-mm serial frozen sections were collected on Superfrost ) Plus microscope slides ŽFisher Scientific.. In situ hybridization was performed as described in Lugo et al. w21x, except that the riboprobes were labeled with w 33PxUTP ŽDuPont NEN. instead of w 35 SxUTP. After overnight hybridization at 558C, the sections were washed as described in w21x. Following RNase A treatment, the final washing condition was with 0.2 = SSC at 658C for 2 h. After dehydration, the sections were first exposed to Hyperfilme-MP autoradiography films ŽAmersham. for 5–6 days followed by NTB-2 emulsion ŽKodak Inc.. coating and stored for 25–30 days at 48C. The coated slides were developed and fixed as instructed by the vendor, then the sections were counterstained with 2% Toluidine blue solution and mounted with Permount ŽFisher Scientific.. In situ hybridization using each specific probe was carried out at least twice and with tissue sections from at least two different mice. 2.4. Image processing Sections were analyzed with a Zeiss Axioplan microscope using either darkfield or brightfield optics. Photographs were transmitted directly into the Roche Image Analysis System ŽRIAS. from a Kontron digital camera attached to the microscope. Composite images were generated using Adobe Photoshop 3.0 ŽAdobe Systems.. Figures were printed using a Fuji Pictography 3000 printer.

3. Results Expression of GIRK 1, GIRK 2, and GIRK 3 transcripts Žsee Table 1, Table 2, and Table 3, respectively. were examined at embryonic day 15.5 ŽE15.5., the day of birth ŽP0., postnatal day 10 ŽP10. and P20 using in situ hybridization. In order to compare the expression levels of these messages during development, all of the tissue slides were processed in parallel, the size of the riboprobes used for the in situ hybridization were similar to each other, and each had an identical specific activity Žsee Section 2: Materials and methods.. In addition, the same exposure time to emulsion and X-ray film was used for all three probes at all ages. For this reason, some regions that showed very low levels of expression may have been recorded as negative. However, using these methods, we feel that we were better able to determine the relative levels of mRNA for each of the GIRK channels. For the most part, each of the three GIRK s appear to be developmentally regulated. For example, the message levels of all three genes were upregulated from E15.5 to P10 in most of the areas that express the genes. These levels of

253

expression were then either sustained or decreased in the P20 brain Žsee Tables 1–3.. Comparatively, GIRK 3 becomes the predominant inward rectifying channel in the brain of older animals. All three GIRK messages showed wide distribution in the developing brain. GIRK 1 showed the least differential expression, both temporally and spatially, and was generally shown to be the lowest overall expressing GIRK-family member in the CNS ŽTable 1.. GIRK 2 and GIRK 3 demonstrated more significant changes in several areas of the brain. In addition, in certain areas of the differentiating diencephalon, GIRK 2 and GIRK 3 messages had opposing temporal distributions ŽFig. 2; Tables 2 and 3.. While the overall expression levels of each of the three transcripts were different, many individual nuclei showed similar levels of expression of all three GIRK s. Conversely, a number of regions that showed differential expression of these three gene transcripts have also been found. These regions include the olfactory bulb, hippocampus, thalamus, substantia nigra and cerebellum. 3.1. Olfactory bulb In the olfactory bulb, GIRK 1 transcripts were first detected at very low levels in the mitral cell layerŽs. at E15.5. At P0, expression levels were slightly increased in the mitral cells, while very low levels of expression became apparent in the anterior olfactory nucleus ŽAON., internal plexiform layer ŽIPL. cells and to a slightly higher degree in the periglomerular cells. Expression levels of GIRK 1 were basically maintained from P0 to P10. At P20, there was a slight increase in expression in the mitral and granule cell populations that are maintained at low levels through P20 ŽTable 1.. At E15.5, GIRK 2 demonstrates the highest expression levels in the olfactory bulb of the three GIRK s. This GIRK 2 mRNA expression was primarily localized to the mitral cells. At P0, very low levels of expression were detected in the AON and IPL cells. Except for the mitral and IPL cells, which maintained their expression levels from P0 to P20, there was a slight and progressive increase in GIRK 2 expression from P0 to P20 ŽTable 2.. Low expression levels of GIRK 3 message were found in most cells of the olfactory bulb at E15.5–P0. By P10, expression levels of GIRK 3 had significantly increased in most of the olfactory bulb’s neuronal populations ŽTable 3.. These increased levels were then maintained through P20. 3.2. Cortex In the cerebral cortex ŽFig. 1A–D., expression of GIRK 1–3 were differentially regulated. At P0, GIRK 1 demonstrated the highest level of expression ŽFig. 1B.. GIRK 3 mRNA, however, also showed low expression levels ŽFig. 1D.. GIRK 2 transcripts, at this time, were

254

Table 1 Developmental expression of GIRK 1 in the CNS CNS region

P0

P10

P20

" " n.a. n.a. n.a.

q " " " q

q q q " "

qq qq q y q

" " " " " n.a. n.a. " " " "

q q q q q q " " q " "

qq qq qq qq qq qq qq qq qq q "

qqq qq qq qq qq q qq " qq " "

" "

" "

" y

y y

y y n.a. y y y " y y

" " " " " q " q y

q " q y y q " q y

q y " y y " y " y

CNS region

E15

Thalamus ant. tier thalamus " vent. tier thalamus " lat. tier thalamus " parafascicular nucleus " post. thalamic nucleus " paraventricular thal. " reuniens nucleus n.a. reticular thalamus y med. geniculate " lat. geniculate " medial mammillary nucleus" sup. mammillary nucleus " Hypothalamus ant. hypothalamus " post. hypothalamus " arcuate hypothal. " DM hypothalamus " VM hypothalamus " PVN hypothalamus " median eminence y preoptic area " Pituitary gland " Midbrain and brainstem IPN y locus coeruleus n.a. red nucleus y lateral lemniscus n.a. sup. colliculus " inf. colliculus n.a. inferior olive y

P0

P10

P20

" " " " " " " q " " " "

q q q " q " q " " q " "

q q q " q " q " " " " "

" " " " " " " " "

" y " q " q " q n.a.

" y " " " y y " n.a.

qq " q q " q y

y " q qq q qq y

y y y " q q y

CNS region Midbrain and brainstem pontine nucleus vent. tegmental area D. tegmental nucleus raphe nucleus central gray med. vestibular nucleus lat. vestibular nucleus Reticular nucleus Cerebellum EGL IGL Purkinje cells nuclear cells molecular layer cell choroid plexus

E15

P0

P10

P20

" " n.a. " " " " "

q q " q " q q q

qq " q " " q q q

q q " " " q q q

" n.a. " n.a. n.a. y

" n.a. " " n.a. y

qq qq q q " y

n.a. q q " " y

n.a., not available; y, no expression; ", very low expression; q, low expression; qq, moderate expression; qqq, high expression; qqqq, very high expression.

S.-C. Chen et al.r Brain Research 778 (1997) 251–264

Olfactory bulb mitral cells granule cells anterior olf. nucleus internal plexiform cells periglomerular cells Telencephalonrlimbic system piriform cortex neocortex hippocampus CA1 hippocampus CA2 hippocampus CA3 hippocampus CA4 dentate gyrus subiculum amygdala lateral septal nucleus med. septal nucleus Basal ganglion caudatoputamen substantia nigra Diencephalon tenia tecta VLDBrHLDB indusium griseum SVZ Ždiencephalon. nucleus accumbens dorsal endopiriform habenular nucleus zona incerta BN stria terminalis

E15

Table 2 Developmental expression of GIRK 2 in the CNS CNS region

P0

P10

P20

q y n.a. n.a. n.a.

q y " " q

" qq q " q

" qq q " qq

q y " " " y n.a. y " " "

qq " q q q q y " q q "

qqq qqq qq qq qq qq qq qq q q "

qq qqq qqq qqq qqq qq qqq qq q " "

" "

y qq

y qqq

y qqq

y y n.a. y y y y y y

" " q y y q " y q

y qq qq y y qq q q y

" q qq y y qq q " y

CNS region

E15

Thalamus ant. tier thalamus qq vent. tier thalamus qq lat. tier thalamus qq parafascicular nucleus " post. thalamic nucleus " paraventricular thal y reuniens nucleus n.a. reticular thalamus " med. geniculate " lat. geniculate " Hypothalamus ant. hypothalamus " post. hypothalamus " arcuate hypothal. y DM hypothalamus " medial mammillary nucleus " sup. mammillary nucleus " VM hypothalamus " PVN hypothalamus " median eminence " preoptic area " Pituitary gland q Midbrain and brainstem IPN " locus coeruleus n.a. red nucleus y lateral lemniscus n.a. sup. colliculus " inf. colliculus n.a. D. tegmental nucleus n.a.

P0

P10

P20

qqq qqq qqq " " " qq q q qqq

qq qqq qq qq qq q qq q qq qqq

q q q q q " q y q q

q q " q q q q " " q qq

" q y q qq qq " " y y n.a.

" " q q q q q qq y y n.a.

" q y q q q q

qq y " " q q q

" y y " q q "

CNS region Midbrain and brainstem vent. tegmental area pontine nucleus raphe nucleus central gray med. vestibular nucleus lat. vestibular nucleus inferior olive brainstem reticular nucleus Cerebellum EGL IGL Purkinje cells nuclear cells molecular layer cell choroid plexus

E15

P0

P10

P20

y q " y q " y q

qq qq q q q " " qq

qq qq q q q " y qq

" qqq " " " " y q

qq n.a. " n.a. n.a. y

qq n.a. qq " n.a. y

qqq qqq qq y " y

n.a. qqq y y y y

S.-C. Chen et al.r Brain Research 778 (1997) 251–264

Olfactory bulb mitral cells granule cells anterior olf. nucleus internal plexiform cells periglomerular cells Telencephalonrlimbic system piriform cortex neocortex hippocampus CA1 hippocampus CA2 hippocampus CA3 hippocampus CA4 dentate gyrus subiculum amygdala lateral septal nucleus med. septal nucleus Basal ganglion caudatoputamen substantia nigra Diencephalon tenia tecta VLDBrHLDB indusium griseum SVZ Ždiencephalon. nucleus accumbens dorsal endopiriform habenular nucleus zona incerta BN stria terminalis

E15

n.a., not available; y, no expression; ", very low expression; q, low expression; qq, moderate expression; qqq, high expression; qqqq, very high expression.

255

256

Table 3 Developmental expression of GIRK 3 in the CNS CNS region

P0

P10

P20

" y n.a. n.a. n.a.

" y " y "

qq qq q y q

qq q qq y q

" " y y y y n.a. " " " "

qq q " " " " " " q " y

qqq qqqq qqqq qqqq qq qq qqq qqq qqq " "

qqq qqq qq qq q " qq qqq qqq q q

" y

" q

q q

" q

y " n.a. y y y y y "

" " y " y " y " "

q " y y q qqq qq qq "

" q y y " qqq qq qq q

CNS region

E15

Thalamus ant. tier thalamus " vent. tier thalamus q lat. tier thalamus q parafascicular nucleus y post. thalamic nucleus " paraventricular thal " reuniens nucleus n.a. reticular thalamus y med. geniculate " lat. geniculate " Hypothalamus ant. hypothalamus " post. hypothalamus y arcuate hypothal. y DM hypothalamus " medial mammillary nucleus y sup. mammillary nucleusy VM hypothalamus " PVN hypothalamus " median eminence y preoptic area " Pituitary gland y Midbrain and brainstem IPN y locus coeruleus n.a. red nucleus y lateral lemniscus n.a. sup. colliculus y inf. colliculus n.a. D. tegmental nucleus n.a.

P0

P10

P20

q qq qq " q " " " " "

qq qqqq qqqq qqq qqqq qqq qq qq qq qqq

qqqq qqqq qqqq qqqq qqqq qqqq qqqq qqqq qq qq

q q " q " q q q " q "

qq qq qq q qq qq q q y q n.a.

qq q qq q q " qq qq y q n.a.

" q y q q " q

qqq q q qqq q qq qq

qq qq q qq qqq qqq qq

CNS region Midbrain and brainstem vent. tegmental nucleus pontine nucleus raphe nucleus central gray med. vestibular nucleus lat. vestibular nucleus inferior olive brainstem reticular nucleus Cerebellum EGL IGL Purkinje cells nuclear cells molecular layer cell choroid plexus

E15

P0

P10

P20

y " y y " y y "

q " " q q q q "

q qqq " q qq q qqqq "

" qqq q qq qqq qq qqqq qq

y n.a. " n.a. n.a. y

y n.a. q " n.a. y

y qq qq q q y

n.a. qqqq qqq qq q y

n.a., not available; y, no expression; ", very low expression; q, low expression; qq, moderate expression; qqq, high expression; qqqq, very high expression.

S.-C. Chen et al.r Brain Research 778 (1997) 251–264

Olfactory bulb mitral cells granule cells anterior olf. nucleus internal plexiform cells periglomerular cells Telencephalonrlimbic system piriform cortex neocortex hippocampus CA1 hippocampus CA2 hippocampus CA3 hippocampus CA4 dentate gyrus subiculum amygdala lateral septal nucleus med. septal nucleus Basal ganglion caudatoputamen substantia nigra Diencephalon tenia tecta VLDBrHLDB indusium griseum SVZ Ždiencephalon. nucleus accumbens dorsal endopiriform habenular nucleus zona incerta BN stria terminalis

E15

S.-C. Chen et al.r Brain Research 778 (1997) 251–264 257

Fig. 1. Developmental expression of GIRK 1–3 in the cerebral cortex. These low-power photomicrographs show GIRK expression at P0 ŽA–D., P10 ŽE–H. and P20 ŽI–L.. A: low-power photomicrograph of Nissl-stained section through the cortex of a P0 mouse at the level of the retrosplenial granular cortex Žctx. and the indusium griseum Žig, arrow.. B: at P0, expression of GIRK 1 is seen in each of the neuronal layers ŽII–VI., but is not seen in the outer molecular layer ŽI.. C: virtually no GIRK 2 expression is seen in the P0 cortex, but is present in the indusium griseum. D: low levels of GIRK 3 expression are seen in all of the cerebral cortical layers ŽI–VI.. E: low-power photomicrograph of Nissl-stained section through the cortex of a P10 mouse at the level of the retrosplenial granular cortex, indusium griseum Žig, arrow. and caudatoputamen Žcp.. F: expression of GIRK 1 is seen at uniform levels in layers II–VI. Low levels of expression are seen in the caudatoputamen. G: expression of GIRK 2 is upregulated compared to P0 in all of the neuronal layers of the cerebral cortex ŽII–VI., and remains high in the indusium griseum. Virtually no expression is seen in the caudatoputamen. H: expression of GIRK 3 is tremendously upregulated compared to P0, and has become the predominant GIRK expressed in the cortex at this time. Low levels of message are seen in the caudatoputamen. I: low-power photomicrograph of Nissl-stained section through the cortex of a P20 mouse at the level of the retrosplenial granular cortex and caudatoputamen Žcp.. J: at P20, the developmental expression of GIRK 1 has remained at a fairly constant level throughout development in both the cortex and caudatoputamen. K: at P20, expression of GIRK 2 has again increased from the levels seen at P10 in all of the cortical neuronal layers. Little expression is still detected in the caudatoputamen. L: the levels of GIRK 3 mRNA remain high at P20 and it still remains the predominant GIRK that is expressed. However, there appears to be a down-regulation of expression in the caudatoputamen, compared to the levels seen at P10. Scale bars: A–D, 450 mm; E–L, 125 mm.

258

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virtually absent. By P10 ŽFig. 1E–H., there was a general upregulation of GIRK expression. At this time, GIRK 3 became the predominant mRNA species ŽFig. 1H., followed in expression levels by GIRK 2, then GIRK 1 expression ŽFig. 1F,G, respectively.. At P20, GIRK 1 mRNA expression is virtually unchanged compared to its levels at P10, while GIRK 2 levels were slightly increased ŽFig. 1K.. At P20, GIRK 3 remained the predominantly expressed inward rectifying channel at this age ŽFig. 1L.. 3.3. Hippocampus As shown in Fig. 2, only very low levels of expression of the GIRK 1 and GIRK 2 messages were detected in the developing hippocampus at E15.5. No expression of GIRK 3, however, was detected. GIRK 1 expression levels were slightly increased at P0 in all of the developing pyramidal cell subregions ŽFig. 2F. with very little expression detected in the developing dentate gyrus. GIRK 1 expression reached its highest developmental level at P10, showing equal levels of expression in all pyramidal cell regions ŽCA1–CA4. as well as in the granule cells of the dentate gyrus ŽFig. 2J.. Expression levels in these subregions were maintained through P20 ŽFig. 2N; Table 1.. GIRK 2 mRNA was also first observed at very low levels by E15.5 ŽFig. 2C.. By P0, expression levels of

GIRK 2, increased slightly and were detected in each of the pyramidal cell subregions, with higher expression levels in CA1 and CA3 ŽFig. 2G.. From P0 to P20, GIRK 2 expression was progressively upregulated in all pyramidal cell regions ŽFig. 2G,K,O; Table 2.. Expression levels of GIRK 3 message in the hippocampus was minimal at both E15.5 and P0 ŽFig. 2D,H.. However, a large increase in its expression level was detected at P10 with the most intense radiolabeling found in the CA1 and CA2 subregions. At P20, the level of GIRK 3 expression was slightly reduced ŽFig. 2P; Table 3.. 3.4. Diencephalon In the diencephalon, the primary area that showed GIRK expression was the thalamus. Differential expression in various thalamic nuclei was detected with probes directed against GIRK 2 and GIRK 3; while GIRK 1 appeared at very low levels throughout all of the thalamic nuclei ŽFig. 2B,F,J,N.. Developmentally, in the E15.5 thalamus, GIRK 2 expression was primarily found in the nuclei of the anterior, ventral and lateral tier nuclei ŽFig. 2A,C.. A similar pattern of expression was observed at P0 ŽFig. 2E,G., although in general, expression levels of GIRK 2 were higher than E15.5. Of the three nuclei in the anterior nuclear group,

Fig. 2. Developmental expression of GIRK 1-3 in the developing forebrain and diencephalon. A: low-power photomicrograph of Nissl-stained section through the diencephalon at E15.5. Several structures are evident, including the anterior thalamic tier of nuclei Žant. thal., the ventricular epithelium of the developing basal ganglion ŽBG., and the developing hippocampus Žhippo.. B: expression of GIRK 1 at E15.5 in the forebrain. At this time, very low levels of GIRK 1 are seen in this region. C: expression of GIRK 2 at E15.5. At this time, there is a generalized low level of expression throughout much of the cortical and diencephalic areas. In addition, high levels of GIRK 2 mRNA are detected in the anterior thalamic nucleus. D: expression of GIRK 3 at E15.5 in the forebrain. At this time, very low levels of GIRK 3 mRNA are seen in this region. E: low-power photomicrograph of Nissl-stained section through the diencephalon at P0. At this time, more definition is present in the forebrain. In this section, the hippocampus Žhippo. has differentiated into a distinct pyramidal layer and dentate gyrus. In addition, the anteriorrlateral thalamus, the lateral dorsal ŽLD., ventral anterolateral ŽVAL., ventral posterolateralrventral medial ŽVPLrM., anteromedial ŽAM. and medial habenular nuclei ŽMeH. are present. F: expression of GIRK 1 at P0 in the forebrain. Low levels of GIRK 1 message are detected in the pyramidal cell layer of the hippocampus, with virtually no expression detected in the dentate gyrus. There is a generalized extremely low level of expression of GIRK 1 that is present throughout the cerebral cortex and thalamic nuclei at this level. G: expression of GIRK 2 at P0 in the forebrain. At this time, high levels of GIRK 2 mRNA are detected in the pyramidal cell layer of the hippocampus as well as throughout the whole of the anteriorrlateral tier of the thalamus. Lower, albeit significant, levels of expression are noted in the medial habenular nuclei as well as in the hypothalamic nuclei that lie ventral to the thalamus. H: expression of GIRK 3 at P0 in the forebrain. Low levels of expression of GIRK 3 are seen at this level in all of the developing forebrain structures. I: low-power photomicrograph of Nissl-stained section through the diencephalon at P10. At this time, the pyramidal cells in the hippocampus have segregated into clearly identifiable regions Žca1–4.. The granule cells have increased in number so that a well-defined dentate gyrus is also present Ždg.. The thalamic nuclei present at this level include the lateral dorsal ŽLD., ventral anterolateral ŽVAL., ventral posterolateralrventral medial ŽVPLrM., mediodorsal ŽMD. and medial habenular nuclei ŽMeH.. J: expression of GIRK 1 at P10 in the forebrain. At this time, low levels of expression are seen in the pyramidal and granule cell layers of the hippocampus. In the thalamus, low levels of expression are detected in the VPMrL and LD nucleus. K: expression of GIRK 2 at P10 in the forebrain. Moderate levels of GIRK 2 mRNA are detected in the hippocampus as well as each of the anterior and lateral tier thalamic nuclei; both of which appear downregulated compared to P0 Žcompare K to G.. Expression of GIRK 2 in the hypothalamus, detected at low levels at P0, is absent. L: expression of GIRK 3 at P10 in the forebrain. Moderate-to-high levels of GIRK 3 mRNA are detected in the cerebral cortex as well as in the CA1 and CA2 regions of the hippocampus. Lower levels of expression are detected in the other hippocampal pyramidal regions as well as in the dentate gyrus. In the diencephalon, there is a generalized upregulation of GIRK 3 expression compared to expression levels seen at P0 Žcompare L to H.. M: low-power photomicrograph of Nissl-stained section through the diencephalon at P20. At this time, the pyramidal cells in the hippocampus have segregated into clearly identifiable regions Žca1–4.. The granule cells have increased in number so that a well-defined dentate gyrus is also present Ždg.. The thalamic nuclei present at this level include the lateral dorsal ŽLD., ventral anterolateral ŽVAL., ventral posterolateralrventral medial ŽVPLrM., mediodorsal ŽMD. and medial habenular nuclei ŽMeH.. N: expression of GIRK 1 at P20 in the forebrain. At this time, low levels of expression are seen in the pyramidal and granule cell layers of the hippocampus. The thalamic expression seen at earlier times has almost totally disappeared. O: expression of GIRK 2 at P20 in the forebrain. High levels of mRNA expression are seen in the pyramidal and granule cell layers of the hippocampus. P: expression of GIRK 3 at P20 in the forebrain. Moderate levels of GIRK 3 mRNA are seen in the pyramidal and granule cells of the hippocampus, although higher levels of expression are seen in the CA1 versus the CA2 or CA3 regions. High levels of uniform expression of GIRK 3 is seen throughout the thalamus. Scale bars: ŽA–P. 1100 mm.

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260 S.-C. Chen et al.r Brain Research 778 (1997) 251–264 Fig. 3. Expression of GIRK 1–3 in developing substantia nigra pars compacta ŽSNpc. and caudal hippocampus. A: P0 substantia nigra stained with Toluidine blue. B–D: expression of GIRK 1, GIRK 2 and GIRK 3, respectively, in consecutive sections of a P0 substantia nigra. High levels of GIRK 2 mRNA expression is seen ŽC. in the SNpc Žarrow.. Compared to GIRK 2, expression of GIRK 1 ŽB. and GIRK 3 ŽD. are seen at relatively low levels in the coalescing pars compacta neurons. E: P10 substantia nigra stained with Toluidine blue. In this section, the developing caudal hippocampus Žhippo. is also visible. F–H: expression of GIRK 1, GIRK 2 and GIRK 3, respectively, in consecutive sections of a P10 substantia nigra. By P10, no expression of GIRK 1 is seen in the SNpc. Low-to-moderate levels of GIRK 1 are seen in the pyramidal and granule cells of the hippocampal formation ŽF.. GIRK 2 is expressed at high levels in the SNpc Žarrow. as well as in the hippocampal pyramidal cells of Ammon’s horn and the granule cells of the developing dentate gyrus. Low levels of GIRK 3 expression are seen in the SNpc and hippocampal formation. I: P20 substantia nigra stained with Toluidine blue. J–L: expression of GIRK 1, GIRK 2 and GIRK 3, respectively, in consecutive sections of a P20 substantia nigra. Like P10, no expression of GIRK 1 mRNA was detected in the SNpc. Low levels continue to be observed in the hippocampus Žhippo.. At P20, GIRK 2 mRNA is still expressed at high levels in the SNpc Žarrow. and hippocampal formation. Low levels of GIRK 3 expression are detected in the SNpc and hippocampus. Scale bars: A–D, 300 mm; E–L, 600 mm.

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Fig. 4. Expression of GIRK 1–3 in the developing cerebellum. A: P0 midline cerebellum stained with Toluidine blue. At this time, the cerebellar cortex is organized into an outer external granule layer Žegl., composed of dividing cells destined to become granule cells, an intermediate molecular layer and a multilayered Purkinje cell plate Žpcp.. B–D: expression of GIRK 1, GIRK 2 and GIRK 3, respectively, in consecutive sections of a P0 cerebellum. GIRK 1 expression ŽB. is virtually absent in the P0 cerebellum. At P0, GIRK 2 is expressed in the developing egl and Purkinje cell plate ŽC.. GIRK 3 is expressed, albeit at low levels, in a similar population of cerebellar cells as that of GIRK 2. E: P10 midline cerebellum stained with Toluidine blue. At this time, the cerebellar cortical egl is composed of a layer of outer dividing cells Žmatrix layer. and an inner postmitotic premigratory zone Žmantle layer., an intermediate molecular layer Žml. and a unicellular Purkinje cell layer Žpcl.. Deep to the Purkinje cells is the internal granule cell layer Žigl.. F–H: GIRK 1 expression is seen at low levels in the igl, but remain virtually absent in the mitotically active egl ŽF.. GIRK 2 expression levels are increased at P10 in all three populations of cerebellar cortical neurons Žegl, pcl, and igl. ŽG.. GIRK 3 expression in the cerebellum remains low in all of the cerebellar cortical neurons. I: P20 midline cerebellum stained with Toluidine blue. At this time, the cerebellar cortical egl has disappeared resulting in the superior position of the molecular layer Žml.. The unicellular Purkinje cell layer Žpcl. and internal granule layer Žigl. remain evident. J–L: GIRK 1 expression is virtually absent from the cerebellum, detailing its transient expression in the cerebellum Žcompare Fig. 4, F to J.. GIRK 2 expression remains at high levels in the igl and pcl, although it appears to be reduced compared to the levels seen at P10 Žcompare G to O.. GIRK 3 expression is seen at moderate levels in the igl and pcl. It is evident that by P20, GIRK 3 is expressed at a higher level than at P10 Žcompare H to L.. Scale bars: A–P, 700 mm.

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i.e. anteroventral ŽAV., anterodorsal ŽAD. and anteromedial ŽAM. nuclei, AD showed the highest expression level of GIRK 2. In the ventral tier, higher levels of GIRK 2 message were found in the ventral anterior ŽVA., ventral posterolateral ŽVPL., ventral posteromedial ŽVPM., and ventral lateral ŽVL. nuclei, and in the lateral tier, the lateral dorsal ŽLD. and lateral posterior ŽLP. nuclei demonstrated the highest expression levels. Significant, albeit lower, levels of GIRK 2 expression were also detected in all of the other thalamic nuclei ŽTable 2.. At P10, the overall level of GIRK 2 expression was slightly decreased ŽFig. 2I,K. compared to P0. This progressive downregulation continued through P20, at which time, only low levels of GIRK 2 message were detected ŽFig. 2M,O. GIRK 3 expression in the thalamus was also regulated developmentally. The first significant expression was detected at P0, where low-to-moderate levels of radiolabeling were observed in most of the thalamic nuclei ŽTable 3, Fig. 2E,H.. From P0 through P10, there was a dramatic upregulation of GIRK 3 expression in all of the thalamic nuclei ŽFig. 2I,L.. High levels of GIRK 3 were maintained through P20. GIRK 3 became the predominant inward rectifying potassium channel expressed in these nuclei since there was almost no expression of GIRK 1 and GIRK 2 in the thalamus.

layer and the post-mitotic, premigratory mantle layer. Lower levels of expression were detected in the future region of the IGL as well as in the Purkinje and nuclear cells ŽFig. 4A,C.. At P10, high levels of expression were detected in the EGL, the Purkinje cell layer as well as the IGL. Little signal was detected in the cerebellar nuclear cells. By P20, the vast majority of the EGL cells have migrated through the molecular layer and the IGL is in its adult configuration. At this time, only the cells in the IGL expressed GIRK 2 transcripts. Thus, GIRK 2 message expression was downregulated in the Purkinje and nuclear cell populations of the older animals ŽFig. 4I,K.. GIRK 3 mRNA expression was first observed at very low levels in the Purkinje cell plate of E15.5 mice. By P0, radiolabeling was detected at low levels in the Purkinje and nuclear cell regions. No expression of GIRK 3 was found in the EGL ŽFig. 4A,D.. Through P10, the pattern of low level expression in the Purkinje and nuclear cells, with no expression in the EGL, was maintained ŽFig. 4E,H.. By P10, there was a significant increase in GIRK 3 expression in the IGL ŽFig. 4B,H.. By P20, GIRK 3 became the predominant inward rectifying channel, with very high levels message detected in the IGL. In addition, moderateto-high levels of expression were seen in the molecular layer, Purkinje cells and deep nuclei ŽFig. 4I,L, Table 3..

3.5. Substantia nigra 4. Discussion In the midbrain substantia nigra, expression of GIRK 1 and GIRK 3 were found at low levels from P0 through P20 ŽFig. 3A-L.. At P0, GIRK 2 expression was seen at moderate levels in virtually all of the large cells in the substantia nigra pars compacta ŽFig. 3C.. The levels of GIRK 2 increased from P0 to P10 ŽFig. 3G., where it remained at a stable level through P20 ŽFig. 3K, Tables 1–3.. 3.6. Cerebellum In the cerebellum, expression patterns of each of the GIRK s demonstrated a different developmental picture. GIRK 1 was first detected at very low levels in the Purkinje cell plate at P0. The external granule layer ŽEGL. cells present on the cerebellar surface, however, did not express this transcript ŽFig. 4A,B.. By P10, the cerebellar cortex has four distinct layers, as a significant number of EGL cells have migrated and settled into the deep cortical internal granular layer ŽIGL.. At this time, moderate levels of GIRK 1 expression were observed in the EGL and IGL, with low expression in the Purkinje and molecular layer. By P20, GIRK 1 message appears to be downregulated, and was present only at low levels ŽFig. 4I,J; Table 1.. GIRK 2 message was expressed in the cerebellum as early as E15.5 in the migrating granuloblasts located on the surface of the cerebellum. By P0, expression of GIRK 2 message was found at moderate levels throughout the whole EGL, including both the mitotically active matrix

In the past few years, four distinct mammalian G-protein-gated inward rectifying potassium channels have been isolated w2,11,15–17x. In this paper we describe the developmental pattern of expression of GIRK 1, GIRK 2 and GIRK 3. We did not examine the expression of GIRK4, since it has previously been shown that it was not present in the developing or adult nervous system w12x. The general localization of GIRK mRNAs is in general agreement with previous reports in adult rat and mouse. Our findings, however, of low levels of GIRK 1 and high levels of GIRK 3 in the P20 mouse brain, are different from the pictures of adult expression shown in Karshin et al. w10x and Kobayashi et al. w11x. Several possible factors may explain these differences, including: Ž1. the relative levels reported by Karshin et al. w10x. were in rat where they noted significant differences between expression patterns in mice and rats; Ž2. further dynamic changes in GIRK 1 and GIRK 3 expression may occur between P20 and adulthood; and Ž3. it appears that factors relating to relative levels of their expression were not controlled for in the work of Kobayashi et al. w11x. Finally, the localization of GIRK1 and GIRK2 protein w19x appears similar to our localization of the cognate message. The pattern of GIRK expression is clearly regulated in development. This regulation is not the result of an artifact due to differences in processing and experimental conditions since all of the samples were processed in an identi-

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cal manner at the same time, and were exposed on X-ray film and autoradiographic emulsion for an identical period of time. The strength of hybridization was controlled for in each probe as GC content ranged from 43 to 56%. In addition, all of the tissue sections were exposed to 1 = 10 7 c.p.m.rml of probe. For GIRK 1 and GIRK 2, many neuronal systems are initially positive throughout the brain. This rather promiscuous expression is pared back to become regionalized in the more mature brain. GIRK 3 expression, on the other hand, tends to come up and stay on during development, with no significant paring-back of positive neuronal systems in the maturing brain. GIRK 1 and GIRK 2 have a fairly prominent developmental regulation, suggesting a special role for these components of the inward rectifying channel in development. These same channels also appear to play a role throughout adulthood since it been shown that GIRK1 and GIRK2 heteromultimerize to form physiologically relevant channels w19x. Since many of the defects that occur in the weaver mouse occur during the prenatal and early postnatal period, the relative levels of GIRK1 to GIRK2 may be more important than GIRK2 to GIRK3. With the identification that a mutation in the GIRK 2 gene is responsible for the weaver mutant phenotype, it becomes important to ascribe the cell death seen in this mutant to a cascade of cellular events that flow from the mutated channel protein. A corollary of this effort must be a predictive hypothesis about how certain populations of cells survive this mutant channel. The present study sheds light upon both of these issues. As shown in this and other studies, the expression of GIRK 2 is far more pervasive than the limited populations of cells that show marked death in the weaver mouse: the cerebellar granule and Purkinje cells w26,27x, the dopaminergic cells of the substantia nigra w24,32x and possibly the hippocampal pyramidal cells Žw25x and Smeyne, unpublished research.. In each of these cases, there is a clear developmental profile of cell death. In the cerebellum, weaver granule cells die at the time when EGL cells exit the cell cycle, prior to their movement into the IGL w26x. Examination of GIRK 1–3 expression demonstrates that by far, the predominant inward rectifying potassium channel at the time of cell death is GIRK 2 ŽFig. 4F–H.. Similarly, in the substantia nigra, the dopaminergic cells of the pars compacta only express significant levels of GIRK 2 during their postnatal development; a time when these cells start to die w23x. These findings suggest that the cell death in weaÕer occurs only in those areas that show moderate-to-high levels of GIRK 2, but not significant levels of GIRK 1 or GIRK 3. This explanation fits the recent physiological data from heterologous expression systems that finds a severe defect in GIRK function when only wvGIRK 2 proteins compose the channel compared to the presence of normal GIRK 2 w33x. However, the sole, or predominant, presence of GIRK 2

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cannot alone be the algorithm of cell death in weaver since structures such as the bed nucleus of the stria terminalis and many nuclei in the thalamus might be expected to show severe cell death. As this does not seem to be the case, one may conclude that many factors, such as the timing of GIRK 2 expression, might be an important factor in a cell’s response to the mutant channel protein. For example, all cells of the cerebellar EGL express GIRK 2, but it is only the premigratory postmitotic cells that undergo death. This suggests that factors such as developmental timing may help to mitigate the effects of the weaver mutation. It is also possible that the loss of cells seen in the weaver mouse brain can be modulated by factors that would overcome the change in ion permeability caused by the GIRK 2 mutation. This could be seen in the cell loss of the substantia nigra where, as in Parkinson’s disease, calbindin-positive nigral cells in weaver are selectively resistant to death w5x. Thus, the handling capacity of intracellular calcium may also play an important determinant of cell susceptibility. This is in line with functional studies of weaver cerebellar granule cells w29x, as well as heterologous expression systems w33x that suggest that the unregulated flux of calcium is at the heart of cell death in the weaver mouse. Thus, one hypothetical action of GIRK channels in development would be to modulate, or act in a pushrpull fashion to excitatory events which lead to increased intracellular calcium or cellular differentiation. A nice example of this may be the proposed role for activation of NMDA receptors in the process of cerebellar granule cell migration w14,20x. The role of ionic channels in the development and maintenance of the nervous system has been of interest for some time w9x. Further research into the role that these inward rectifying potassium channels play in the nervous system may lead to a greater understanding of developmental neurodegenerative disorders. In addition, whatever the means that the mutated GIRK 2 genes affects cellular death, the present findings mandate a renewed look at the neuropathology of the weaver mutant mouse. Acknowledgements The authors thank Ms. Te Wang, Mr. J.D. Wallace, and Ms. Jeanine Salerno for technical assistance. R.J.S. is supported in part by NIH Cancer Center Support CORE Grant P30 CA21765 and by the American Lebanese Syrian Associated Charities ŽALSAC.. D.G. is supported in part by the UT Department of Anatomy and Neurobiology and the UT Research Contingency Fund. References w1x A.M. Brown, L. Birnbaumer, Ionic channels and their regulation by G protein subunits, Annu. Rev. Physiol. 52 Ž1990. 197–213. w2x N. Dascal, W. Schreibmayer, N.F. Lim, W. Wang, C. Chavkin, L. DiMagno, C. Labarca, B.L. Kieffer, C. Gaveriaux-Ruff, D.

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