1 Interdepartmental Neuroscience Program and , 3 Department of Ophthalmology and Visual Science and Section of Neurobiology, Yale University School of Medicine, New Haven, CT 06520, USA
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Abstract |
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Introduction |
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Rodent visual cortex serves as a good model for studying the function of CNG channels in both developmental and adult plastic processes, since all of the major events ranging from cell proliferation to process outgrowth and refinement have been well documented in this region of cortex (Angevine and Sidman, 1961Altman, 1966a
,b
; Miller, 1988
; Naegele et al., 1988
; Altman and Bayer, 1989
; Bayer et al., 1991
; Kageyama and Robertson, 1993
). In particular, the generation of the cortical layers and their relationship to the ingrowth of axons from the lateral geniculate nucleus have been well described (Peters and Feldman, 1976
, 1977
, 1979
; Peters and Saladanha, 1976
; Peters et al., 1976
). These events have largely been considered to occur in the absence of activity, while later refinement or final wiring of connections to visual cortex has been thought to depend on neural activity, i.e. to require sodium-mediated action potentials (see e.g. Constantine-Paton et al., 1990
; Goodman and Shatz, 1993
). It is becoming clear, however, that the early events such as migration and process outgrowth may be influenced by a different form of activitythat of calcium fluctuations produced by cyclic nucleotides or other transmitters (Mattson et al., 1988
; Bolsover et al., 1992
; Kater et al., 1994
; Komuro and Rakic, 1996
).
As a first step in determining the possible role of cyclic nucleotide-gated channels in early developmental events, we performed semi-quantitative RT-PCR and in situ hybridization studies to determine the developmental time course and localization of all three CNG family members. We demonstrate that the three channel subtypes are each expressed in a distinct temporal and spatial pattern, with the rod and olfactory subtypes being expressed at higher levels during the time of cell migration and rapid dendritic outgrowth, and the cone/testis subtype being highly expressed after eye opening. These data suggest that CNG channels are present at the right time and place to play an important role in events in cortical development such as cell migration, dendritic outgrowth and synapse formation.
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Materials and Methods |
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The semiquantitative PCR method of Chelly and Kahn (1994) was used after initial studies showed that Northern hybridization was impractical because of the low abundance of the molecules (data not shown). We emphasize that this PCR method gives information about the relative abundance of a given molecule, in this case with respect to age. Intermolecular comparisons cannot be made nor absolute quantities of mRNA determined. To perform a developmental time series, three litters of Long Evans rats were used. Mothers were housed with their litters in a room with a 12 h light/12 h dark cycle, and given food and water ad libitum. For each time point, three pups were taken from one litter, sacrificed by CO2 inhalation and the visual cortices dissected, pooled and immediately frozen in liquid nitrogen for RNA extraction by the one-step Trizol method (Gibco BRL, Gaithersburg, MD). Anatomical boundaries were approximated according to Schober and Winkelmann (1975). For embryonic tissue, the pregnant dam was sacrificed by CO2 inhalation, embryos removed and cerebral cortices dissected from the entire litter (~10 embryos). A 10 µg quantity of total RNA was treated with 40 U of RNase-free DNase (Boehringer-Mannheim, Indianapolis, IN) for 30 min and subsequently used for each random hexamer primed cDNA reaction according to the manufacturer's protocol (Gibco BRL). Lack of genomic DNA contamination was verified by performing PCR with actin primers before reverse transcription. No band was detected. Two and a half microliters of single-stranded cDNA reaction was used in a 50 µl PCR reaction carried out using 25 cycles of 94°C for 1 min, 5256°C for 1 min and 72°C for 1.5 min. Primer pairs used were the following: rod channel 5'-GATATTAAACTAACCATGAAGAC -3' (bp 15+8), 5'-TCAAGTTTAAACTGCAGGTTTG-3' (bp 719
698); olfactory channel 5'-GGATGATGACCGAAAAATCC-3' (bp 1
+18), 5'TGATAGAAGCCACATCCAAATTG-3' (bp 692
668); cone/testis channel 5'-CTGAATGTGACTGTGCAGAGATG-3' (bp 252
274), 5'-CAAGGTCTTTGTGTAATGTTTCCA-3' (bp 834
811); glyceraldehyde-3-phosphate dehydrogenase (G3PD; Baier et al., 1993) 5'-GTGAAGGTCGGAGTCAACG-3' (forward), 5'-GGTGAAGACGCCAGTGGACTC-3' (reverse); ß-actin (5'-GTGGGGCGCCCCAGGCACCA-3' (forward), 5'-CTCCTTATTGTCACGCACGATTT-3' (reverse). Primers for the rod (Barnstable and Wei, 1996
) and olfactory (Dhallan et al., 1990
) subtypes are from rat and those for the cone/testis (C.J. Barnstable, unpublished data) are from mouse. For a given set of primers, the entire time series was processed simultaneously for valid comparison. Primers were chosen from the 5' region of the rod, olfactory and cone/testis channel subtypes after sequence comparison and preliminary testing of primers demonstrating the 5' region to have the lowest degree of homology among subtypes. The specificity of primers was confirmed by testing each primer pair on cDNA clones of each channel subtype. Each set amplified a sequence only from the appropriate channel plasmid (Fig. 1B
). The number of cycles used for PCR was verified to be in the exponential phase of amplification for each channel subtype (Fig. 1A
). In each case, 25 cycles was shown to be well below the plateau range, indicating that intensity of a PCR band is proportional to the starting RNA concentration. The age chosen for each channel subtype in performing the cycle series for determination of linear range of amplification was that exhibiting a high level of mRNA expression by RT-PCR and/or in situ hybridization, i.e. P0 for rod, P7 for olfactory and P15 for cone/testis channels.
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PCR samples were electrophoresed on 1% agarose gels, denatured and neutralized as described (Sambrook et al., 1989) before being transferred overnight onto nylon membranes (Schleicher and Schuell, Keene, NH). Membranes were prehybridized for 2 h at 42°C, 1 x 106 c.p.m./ml of [32P]dCTP random-primed labeled probe was added and hybridization proceeded overnight.
Data Analysis
Autoradiograms were digitized and relative differences in band intensities across ages for each molecule were quantitated using NIH Image gel plotting macros. Graphs were generated on the graphing and statistical program Prism (GraphPad, San Diego, CA). Regression analysis for rod channel data was performed using the same program. The value for each age on a graph represents one reading of one PCR run. In order to account for any variation in efficiency of cDNA synthesis, all transmittance values were normalized to actin by taking the ratio of a given value at a particular age to the actin value at that age [see Ahmad et al. (1990) for use of actin]. The choice of actin was validated by using quantities of plasmid DNA in PCR reactions to simulate cellular concentrations of a high abundance molecule (actin) and a low abundance molecule (rod channel). Actin was in a 100-fold greater concentration. Identical slopes were obtained for the two molecules (Fig. 1C). Further, in PCR reactions, coamplification of actin and G3PD, another control molecule, yielded an identical pattern, suggesting the pattern observed with actin did not reflect an effect of age.
Complete analysis was performed on four independent occasions and one representative data set is shown. Each data point in this set represents mRNA pooled from two or three animals. Combining or averaging data from different sets was invalid due to differences in autoradiograph exposure. The reproducibility of the PCR method, however, can be seen in Figure 4, in which E13/14 and P0 cDNAs were in amplified parallel in three independent PCR runs, electrophoresed on the same gel and Southern blotted. Values at each age were averaged to give the SEM.
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Visual cortices of three animals for each age were pooled, and 40 µg of total RNA obtained from this tissue was used for Northern blotting as described in Sambrook et al. (1989) with the following modifications. Prehybridization was performed in 5x SSC, 0.01 M NaPO4, 0.5 µg/ml salmon sperm DNA, 1x Denhardt's, 5% dextran sulfate, 0.1 mg/ml each of poly U, A and C, and 50% formamide at 42°C for 1/2 h. Hybridization then proceeded overnight with 1 x 106 c.p.m./ml random-primed labeled DNA probe. RNA from the hippocampus of one P55 rat was used as a control, since previous in situ hybridization studies have shown rod channel mRNA to be high in adult hippocampus (Kingston et al., 1996).
In Situ Hybridization
Brains of animals perfused with PBS followed by 4% paraformaldehyde were removed and postfixed for several hours at 4°C. After cryoprotection, 30 µm sagittal sections were cut into 6or 24-well plates containing 2x SSC and then stored at 20°C in prehybridization solution. Digoxigenin-labeled RNA probes were transcribed according to manufacturer's instructions (Boehringer-Mannheim) from linearized PCR II plasmid (Invitrogen) containing the appropriate cloned PCR product. In the case of the cone/testis channel, a 40-fold excess of unlabeled probe was added to the labeled probe due to a single promoter site in the vector such that no sense strand control could be synthesized. Free-floating sections were incubated in prehybridization buffer for 24 h at 50°C before hybridization overnight at 50°C with 250 ng/ml of probe. Washes, antibody incubation and color reaction were as described previously (Wahle and Beckh, 1992). For a given molecule, all ages were processed and color reaction terminated simultaneously such that comparison of the intensity of label among ages reflects relative levels of mRNA. Three to six in situ runs on ~10 sections of each age/run were performed and the results analyzed taking note of label in all brain regions. The focus, however, was the visual cortex.
Determination of Cortical Layers
Ethidium bromide (EtBr), a nuclear stain that intercalates into doublestranded DNA, was used in the determination of cortical layers since formazan salts deposited in the in situ color reaction preclude standard Nissl staining. Sections were counterstained with 0.0004% EtBr after completion of the in situ hybridization and mounted in 50% glycerol.
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Results |
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Quantitation of the RT-PCR results revealed that each channel subtype had a unique pattern of expression in developing cortex (Fig. 3A). For the rod channel, mRNA levels were high at birth (P0). Subsequently, message levels fell rapidly until they were barely detectable by P55. Note that mRNA levels in the hippocampus of P55 rat remained high, as also shown by Northern blot in Figure 3B
. Further, there was strong agreement between RT-PCR and Northern hybridization for this subtype despite a certain degree of noise in the PCR method (e.g. P21). Non-linear regression analysis modeling the data as an exponential decay yielded a very close fit (F = 0.7138; data not shown). The residuals were close to zero, indicating that factors other than noise did not contribute to the unusually large value. Thus it appears that message for the rod CNG channel is both developmentally and regionally regulated. Given the high level of rod CNG channel mRNA at birth (P0), it was of interest to determine whether and at what level it was expressed before birth. cDNA from embryonic day 13/14 cerebral cortex (time of birth of first cortical cells in rat) was thus compared with P0 using RT-PCR as above. The data in Figure 4
show that the level of rod channel mRNA is very low at E13/14, about one-quarter that seen at birth.
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The developmental time course of the cone/testis channel exhibited a bimodal distribution, peaking at P15 (eye opening) and P40 (Fig. 3A). The latter age corresponds to the end of the critical period for plasticity exhibited in response to monocular deprivation (Fagiolini et al., 1994
). Although, strictly speaking, ocular dominance columns are not formed in rodents, as in other mammals, during the critical period axon arbors from LGN neurons undergo refinement in layer IV of visual cortex due to competition between the two eyes.
Localization of the Three Channel Types in Developing Rat Visual Cortex by In Situ Hybridization
Given the time course of each of the CNG channel subtypes, we were interested to know the laminar distribution in the visual cortex during development, and whether there would be differences in the localization pattern not only at different ages for a given CNG channel type but also among types. The P0 cortex showed labeling for the rod CNG channel in the cortical plate, subplate and subventricular zone (Fig. 5A). By P7, label was present in layers II/III and V (Fig. 5B
). It was clear from a comparison between digoxigenin-labeled and ethidium bromide-stained sections that not all neurons in a given layer expressed the rod subtype (data not shown). Label was undetectable by P35 (Fig. 5C
). The sense control, shown inset in Figure 5B
, did not label.
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Regional Specificity
Not all brain regions had a low level of CNG channel expression at later ages. Two such examples are the hippocampal formation and the cerebellum. In the hippocampus at P35, label for the rod, olfactory and cone/testis channel subtypes remained strong in pyramidal cells and dentate granule cells (Fig. 5C,F,I, right insets), a time when label was very low in the cortex. In the cerebellum at this age, all three channels were expressed in Purkinje cells. Intensity of label varied, however, with olfactory channel (Fig. 5F
, left inset), being stronger than either the rod (Fig. 5C
left inset) or cone/testis (Fig. 5I
, left inset) channels.
Lastly, the pattern of expression of CNG channels in the cortex exhibited striking specificity for only the sensorimotor regions both at P0 (Fig. 6A) and P7 (Fig. 6B
). In P7 sections (e.g. Fig. 6B
), the sensorimotor cortex was labeled heavily anteriorly, with two dense bands being apparent and corresponding to layers II/II and V. At the rostral-most extent, there was a hint of a third band corresponding to deep layer V or upper layer VI. A gap in cortical label was then apparent spanning the hippocampus below. This corresponded to parietal cortex. At the caudal-most extent, two intense bands of label were seen once again, corresponding to the occipital region.
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Discussion |
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Although there have been a number of in situ hybridization studies of CNG channel expression (El-Husseini et al., 1995; Kingston et al., 1996
; Bradley et al., 1997
), this is, to our knowledge, the first to detail the developmental expression of CNG channel subtypes in the cortex. Furthermore, the prior studies dealt only with the olfactory channel (El-Husseini et al., 1995
; Bradley et al., 1997
) or the rod and olfactory subtypes (Kingston et al., 1996
). Indeed, Bradley et al. (1997) failed to detect mRNA for the rod CNG channel subtype in any adult brain region tested by RT-PCR. The most plausible explanation for such a discrepancy with our results and those of Bradley et al. (1997) is that, as we have shown, mRNA levels have reached a very low level by adulthood. Last, to our knowledge there are only two studies dealing with expression and localization of the cone/testis CNG channel subtype. The first clearly demonstrated the presence of alternatively spliced mRNAs in chick pineal gland, but no localization was performed (Bonigk et al., 1996
). The second performed in situ hybridization on rat brain, but only detected expression in hypothalamus and pineal gland (Sautter et al., 1997
). It is unclear as to why no labeling in the cortex or other regions of the brain was detected, but this may be due to a difference in methods.
Peters and Feldman (1976) showed that geniculocortical afferents primarily terminate in layer IV and lower layer III of rat visual cortex, and to a small extent, layers I and VI. Importantly a great majority of these synapses are made on basilar dendrites of layer III cells and apical dendrites of layer V and VI pyramidal cells. Although most of the rod and olfactory CNG channel mRNAs were localized to somata and primary dendrites, it is possible that these channel proteins are present on growing dendrites of layer III and V cells and play a role in synapse formation between geniculocortical terminals and layer III and V dendrites. Electron microscopy with channel-specific antibodies must be done, however, to validate such a hypothesis.
With regard to the cone/testis channel, the situation may be more complex. In layer VI, cytoplasmic labeling of cells is quite clear. In contrast, the label in layer IV is rather diffuse and is clearly not cellular. Other in situ hybridization evidence has raised the possibility of mRNA in neuronal processes (Wanner et al., 1997; Bassell et al., 1998
; Landry and Hokfelt, 1998
). Thus the diffuse labeling might be indicative of mRNA in geniculocortical axon tips, or in dendritic processes of layer IV cells.
In the cortex, olfactory channel label is restricted to sensorimotor regions (Fig. 6B). This may indicate a special relationship to thalamic afferents. Indeed, the overall pattern of mRNA expression for this channel subtype strikingly resembles that of Ramón y Cajal's illustration of afferent and efferent pathways to somatic sensory-motor and occipital regions (Ramón y Cajal, 1995). This suggests a possible evolutionary extension of CNG channel function from primary sensory organs such as the eye or olfactory epithelium, to regions of cortex receiving sensory information from these organs via subcortical structures.
The fact that the rod and olfactory subtypes have an overlapping temporal and spatial pattern of expression, and the fact that mRNA levels for both are high at a time early in postnatal development when rapid process outgrowth occurs, suggests that the two may function together or sequentially to fine-tune growth cone responses to environmental signals elevating either cAMP or cGMP. Indeed, Song et al. (1997) have recently shown that the level of cAMP is very important in determining attraction or repulsion of a growth cone. Earlier reports have suggested that cAMP and cGMP have opposite effects on growth cone behavior (Bolsover et al., 1992). CNG channels could participate in such a process, since the rod CNG channel is physiologically activated only by cGMP, while the olfactory CNG channel can be activated by both cAMP and cGMP.
Calcium levels in growth cones are known to be important in regulating growth cone motility. CNG channels are highly permeable to Ca2+, and the ß subunit of the olfactory CNG channel is present in vitro at the tips of hippocampal growth cones (Bradley et al., 1997) and visual cortical processes (D.R. Samanta Roy and C.J. Barnstable, unpublished observations). Thus one may hypothesize that CNG channels, being developmentally regulated and highly expressed during rapid dendritic outgrowth (for rod and olfactory subtypes) may be involved in process outgrowth. Although it remains to be tested in mammals, strong support for such a role has been provided from mutations in two Caenorhabditis elegans CNG channel genes which result in marked abnormalities in axonal outgrowth (Coburn and Bargmann, 1996
).
Our data thus suggest the opportunity for a rich array of responses to second messengers and neurotransmitters in the developing cortex, allowing for fine-tuning of growth cone responses to environmental signals, and point to CNG channels as being present at the right time and place to play an important role in these events. Continued exploration of the functional role of CNG channels in early development should prove enlightening.
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Notes |
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Address correspondence to Deborah R. Samanta Roy, PhD, Section of Neurobiology, Yale University School of Medicine, PO Box 208001, New Haven, CT 06520-8001, USA. Email: dsamanta{at}biomed.med.yale.edu.
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Footnotes |
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References |
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