Neural nicotinic acetylcholine responses in sensory neurons from postnatal rat

248

Brain Research, 533 (1990) 248-254 Elsevier

BRES 16049

Neural nicotinic acetylcholine responses in sensory neurons from postnatal rat Nikolaus J. Sucher, Toni P.O. Cheng and Stuart A. Lipton Department of Neurology, The Children's Hospital, and the Program in Neuroscience, Harvard Medical School, Boston, MA 02115 (U.S.A.) (Accepted 29 May 1990) Key words: Neuronal bungarotoxin; Nicotine; Dorsal root ganglion; Receptor

The whole-cell configuration of the patch-clamp technique was used to study nicotinic acetylcholine (ACh) responses in freshly dissociated dorsal root ganglion (DRG) cells from postnatal rat. At negative holding potentials with physiological solutions in the bath and the pipette, ACh (20/aM), nicotine (5/~M) or DMPP (20 #M) activated inward currents in 51% of the cells. Average current density was higher in 1-month-old compared to newborn animals. Nicotinic agonist-induced currents were unaffected by atropine (10/tM) but reversibly blocked by hexamethonium (20/aM). Although labeling with fluorescent a-bungarotoxin (BGT) demonstrated the presence of toxin binding sites on DRG cells, DMPP-induced inward currents were unaffected by micromolar BGT. Neuronal bungarotoxin (100 nM), in contrast, led to a largely irreversible block of the nicotinic responses. These results show that postnatal DRG cells express functional nicotinic acetylcholine receptors (nAChR) of a neuronal type. INTRODUCTION With the exception of dorsal root ganglion ( D R G ) or spinal ganglion cells, functional nicotinic acetylcholine receptors ( n A C h R ) have been unequivocally identified on the cell surface of all neural crest-derived neurons is. It is not clear whether D R G neurons are cholinoceptive although radioactive nicotine and a-bungarotoxin label the axoplasm of the central and the peripheral processes as well as the cell body of D R G neurons28"31. Moreover, nicotinic receptors have been localized in a subpopulation of rat D R G neurons by immunohistochemical labeling using monoclonal antibodies35. Early electrophysiological findings indicated that m a m m a l i a n D R G neurons did not respond to application of acetylcholine 17. However, recent preliminary evidence suggested the presence of functional n A C h R s in a subpopulation of chick D R G n e u r o n s 3. We used the patch-clamp technique to clarify whether functional n A C h R s are present on the cell body of freshly dissociated D R G neurons from postnatal rats. The snake venom-derived neurotoxins a-bungarotoxin (BGT) and neuronal bungarotoxin (NBT) have been shown to distinguish between skeletal muscle and neuronal types of n A C h R s , respectively24. Neuronal n A C h R s are thought to be composed of at least 2 different subunits, designated as ct and fl, forming a nicotinic receptor gene

family33'37. Sensitivity of n A C h R s to blockage by NBT has been found to be d e p e n d e n t on their specific subunit composition2'8'9't3'36. Therefore, we used these toxins to further characterize the nicotinic receptors found on D R G cells.

MATERIALS AND METHODS Dissociation and cell culture The DRGs were dissected from 2-30-day-old postnatal rats and rinsed several times with Hanks' saline (composition in mM: NaCI, 137; NaHCO 3, 1; Na2HPO4, 0.34; KCI, 5.36; KH2PO4, 0.44; CaC12, 1.25; MgSO4, 0.5; MgC12, 0.5; N-2-hydroxyethylpiperazine-N'-2 ethanesulfonic acid (HEPES)-NaOH, 5; dextrose, 22.2; Phenol Red, 0.001% v/v; adjusted to pH 7.2 with 0.3 M NaOH). Next, the DRGs were trimmed to remove excessive nerve fibers and connective tissue, digested with an enzyme mixture (3/A/ml papain and 0.1 mg/ml collagenase) for 30 min in 2 successive episodes, and then triturated with a Pasteur pipette to dissociate the DRG cells. Dissociated DRG cells were then cultured in Earle's minimum essential growth medium (Gibco, #1090) containing 10% (v/v) heat-inactivated fetal calf serum, 5% (v/v) rat serum, glutamine 2 mM, gentamicin 1 /~g/ml, dextrose 16 mM and 0.7% (w/v) methylcellulose. The cultures were kept for a minimum of 3 h in a humidified chamber with 5% CO2/95% air at 37 °C before they were used for electrophysiological studies or a-bungarotoxin labeling. a-Bungarotoxin labeling Freshly dissociated DRG cell cultures were fixed for 15 min with 4% paraformaldehyde in 0.2 M phosphate buffer at pH 7.3, and then treated with 1 M phosphate-buffered glycine to neutralize unreactive fixative. After repeatedly rinsing with phosphate-buffered saline (PBS), the fixed cultures were incubated with phy-

Correspondence: S.A. Lipton, Department of Neurology, The Children's Hospital, Enders Building, Room 350, 300 Longwood Avenue, Boston, MA 02115, U.S.A. 0006-8993/90/$03.50 (~ 1990 Elsevier Science Publishers B.V. (Biomedical Division)

249 coerythrine-a-bungarotoxin conjugate (1.5 gg/ml) for 1 h. For controls the cultures were preincubated with 200/~M acetylcholine, 100/~M nicotine or 2.5/~M unlabeled a-bungarotoxin in PBS prior to labeling with the fluorescent marker. The labeled cultures and the controls were wet-mounted for fluorescence light microscopy, using a Zeiss-IM 35 inverted microscope equipped with a 63 × neofluor objective.

Electrophysiological recordings Patch-clamp recordings1°As were performed 1'2°'2~ at room temperature 3-7 h after plating. Patch pipettes were pulled in a 2-stage process on a BB-CH puller, coated with Sylgard, and fire-polished in a microforge. The electrode resistance measured in the bath was 1-4 MI2. An indifferent Ag/AgCl electrode was connected to the bathing fluid via an agarose bridge with 2 M KCI. Whole-cell recordings were made only from spatially compact cells (lacking processes) to ensure an adequate voltage clamp. Membrane capacitance and input resistance were monitored. Series resistance values were less than 10 MI2 and, if necessary, electronically corrected with the series resistance compensation circuit of an EPC-7 patch clamp amplifier (List Electronic, Darmstadt, ER.G.). Monitoring of the activation of voltage-gated sodium currents by depolarizing voltage steps was used to check the quality of the whole-cell voltage-clamp before recording drug-induced responses.

Data acquisition and analysis The membrane currents were recorded with a patch clamp amplifier, digitized with a 12-bit, 125-kHz analog to digital converter (Model DT2782 DMA: Data Translation), and viewed both on an analog oscilloscope (Model 5111A, Tektronix) and on a digital display (Model 1345A, Hewlett-Packard). The sampling rate was varied from 100 gs to 4 ms, and the signals were filtered at 0.5-5 kHz (Model 4302, Ithaco, with a Bessel frequency cut-off characteristic of 48 dB/octave). Holding and command potentials were generated by a PDP-11/23 computer (Digital Equipment Corporation) and a digital-to-analog converter (Cheshire Data). Data were stored on a 30 megabyte Winchester disk (Data System Design, U.S.A.).

RESULTS Using the whole-cell configuration of the patch-clamp technique 15, electrophysiological recordings were obtained from 47 dorsal root ganglion cells. Freshly dissociated D R G cells were uniformly round, had a smooth, phase-bright appearance, and had no or only scarce neuritic processes. The soma diameter of the cells varied between 18 and 50/~m. The average size of the D R G cells was 26.5 + 5 . 5 / t m (mean + S.D., n = 17) and 37.4 + 8.2 /~m (mean + S.D.; n = 10) in P2-5- (postnatal days 2-5) and 1-month-old (P30) animals, respectively. U n d e r current-clamp mode and with a NaCI solution in the bath and KC1 in the pipette (see Materials and Methods section), the cells m a i n t a i n e d a resting membrane potential o f - 6 3 . 0 + 6.1 m V ( m e a n + S.D.; n = 5). Input resistance values of the D R G cells recorded with the patch-pipettes were 914 + 271 MS"2 (mean + S.E.M.; n = 18) when the patch pipette contained the CsCI/TEA-CI solution and 331 + 105 MI2 (mean + S.E.M.; n = 11) with the KCI solution. Average membrane capacitance was 1.11 + 0.14~F/cm 2 in P2/3 (mean + S.D.; n = 9) cells and 1.36 + 0.23/~F/cm2 in P30 (mean + S.D.; n = 8) cells. Additional recordings obtained from 42 cells cultured for 1 day or longer yielded essentially similar results compared to those reported here for freshly dissociated cells. The cells showed outgrowth of neurites within one day in culture. The younger cells (P2) grew more extensive neurites compared to P30 cells.

Solutions Prior to each experiment, the culture medium was replaced by Hank's saline (see above) containing 2.5 mM calcium. A stainlesssteel insert was placed into the culture dish to limit the fluid volume to approximately 100/~1. The cells were continuously superfused at a rate of ~0.8 ml/min. For whole-cellrecording the pipettes were filled with a KCI solution containing in mM: KCI, 140; MgCl2, 2; CaCl2, 1; ethyleneglycol-bis-(fl-aminoethylether)tetra-acetate(EGTA), 1.5-3; HEPES-NaOH, 10; adjusted to pH 7.2 with 0.3 M NaOH. In some experiments the KCI was replaced by 120 mM CsCI and 20 mM tetraethylammonium (TEA)CI to block potassium currents 2°. The internal free calcium concentration of the solutions was calculated to be 4 × 10 -7 M for 1 mM CaCI2 with 1.5 mM EGTA and 1.6 x 10 -7 M for 1 mM CaCl2 with 2.25 mM EGTA4.

Drugs Drugs were dissolved in Hanks' saline and applied by pressure ejection through micropipettes (5-10 am tip diameter) positioned at a distance of about 10-20 /~m from the cell under study. The concentrations of the drugs applied by pressure ejection are those in the micropipette and represent an upper limit for the actual concentration attained at the cell membrane due to dilution with the bathing fluid upon ejection. BGT and NBT, dissolved in Hanks' saline, were superfused. For superfusion of the toxins, and subsequent washout, the solutions were switched with a dead time of approximately 1 min1AS, Fluorescently labeled a-bungarotoxin was obtained from Molecular Probes (Eugene, OR). Acetylcholine, hexamethonium, nicotine, and dimethylphenylpiperazinium (DMPP) were obtained from Sigma Chemical Co (St. Louis, MO). Neuronal bungarotoxin (NBT) and unlabeled a-bungarotoxin (BGT) were a kind gift of Dr. R. Loring, Northeastern University, Boston.

Effects of nicotinic agonists and antagonists on whole-cell currents Responses to nicotinic agonists were obtained in 51% of all D R G cells tested (n = 47). Acetylcholine (20 # M ) induced inward currents between - 5 and - 4 0 p A at a holding potential VH = - 6 0 mV. A C h - i n d u c e d currents were unaffected by the muscarinic antagonist atropine (10/~M) when present in the superfusion medium. Low micromolar doses of nicotine (5/~M) and D M P P (20/~M), a selective agonist at n A C h R s , induced inward currents of similar size when compared to A C h in single cells. Full d o s e - r e s p o n s e curves were not obtained, however. Agonist-induced inward currents were reversibly blocked by concomitant administration of the ganglionic blocking drug h e x a m e t h o n i u m (20 /~M). Typical examples of responses to these drugs are shown in Fig. 1. The percentage of cells with nicotinic responses was found to be similar, when recordings made from the younger animals (P2/3; 60% responding, n = 10) were compared to those in the older animals (P30, 70% responding, n = 7). H o w e v e r , the average current density was increased more t h a n 2-fold when the responses in P 2 - 7 cells were compared to P30 D R G cells

250

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20 IJM ACh + 10 MM ATR

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Fig. ]. Nicotinic agonists induce inward currents in freshly dissociated DRG cells from postnatal rat (P4 and P5). A: whole-cell recordings were obtained with a patch-pipette from a freshly dissociated DRG cell from rat (P4) voltage-clamped at -60 inV. Acetylcholine (20/~M) and nicotine (5/~M) induced inward currents at physiological holding potentials. The ACh-response is unaffected by the presence of the muscafinic antagonist atropine (ATR; ]0/~M). The drugs were dissolved in Hanks' saline and microperfused onto the cells from a glass pipette by pressure ejection for the time indicated by the dashed line above the traces. The pipette contained a CsC] internal solution (see Materials and Methods section for composition). B: responses of another freshly dissociated DRG cell from rat (PS) to the nicotinic agonist DMPP (20 #M). The response was completely and reversibly blocked by concomitant administration of the ganglionic blocking drug hexamethonium (20/~M). The cell was voltage-clamped at -80 inV. External and internal solutions same as in A. A small part of the control trace exceeded the range of the analog-to-digital converter at this gain and was slightly clipped.

20 MM OMPP (--- Hanks (control) (-- 5 min o(BGT <-- 4 min wash (--- 3 min NBT ~

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Fig. 2. Nicotinic responses in freshly dissociated DRG cells from postnatal rat are blocked by neuronal bungarotoxin (NBT) but not a-bungarotoxin (aBGT). Whole-cell patch-clamp recordings were obtained from a freshly dissociated DRG cell from rat (PS) voltage clamped at -80 inV. The pipette contained a CsCI internal solution (see Materials and Methods for composition). The nicotinic agonist DMPP was microperfused onto the cell from a glass pipette by pressure ejection for the time indicated by the dashed line above the traces. DMPP induced an inward current when the cell was superfused with Hanks' saline (upper trace). The DMPP-induced response was only slightly smaller after having superfused the cell for 5 min with the neuromuscular blocking drug a-bungarotoxin (l gM; 2nd trace). After another control response was obtained following"a 4-min wash (3rd trace), neuronal bungarotoxin (100 nM) added to Hanks' saline was superfused on to the cell. The DMPP induced response was almost completely blocked after 3 min of perfusion (4th trace). The NBT block was largely irreversible (lowermost trace) and persisted for the duration of the recording (75 min).

(0.83 pA/cm 2 vs. 1.97/~A]cm 2, respectively). C o m p a r i n g all responding and n o n - r e s p o n d i n g cells, no clear correlation with cell size was evident, except that cells with a d i a m e t e r below 25/~m were never found to r e s p o n d to nicotinic agonists. Nicotinic agonist-induced responses in single cells were recorded for m o r e than 1 h. T h e responses to low micromolar doses of agonist were usually stable, but n e e d e d - 2 min for complete recovery b e t w e e n r e p e a t e d short ( ~ 1 0 s) applications of the drugs. High agonist doses (100 # M applied for - 1 0 s) could abolish all nicotinic responses in single cells for m o r e than 10 min. The current induced by higher concentrations of D M P P (50 or 100 a M ) desensitized with time constants between 1 and - 1 0 s varying from cell to cell (internal free Ca2+-concentration was ~ 1 0 -7 # M , the external solution contained 2.5 m M Ca2+).

Effects of a-bungarotoxin and neuronal bungarotoxin Functional n A C h R s have b e e n reconstituted in frog oocytes by injecting different combinations of a- and fl-subunit m R N A . Susceptibility to and degree of blockage by N B T of A C h R s have been shown to be d e p e n d e n t on their subunit composition 9. Nicotinic receptors on autonomic ganglion cells 14, rat sympathetic neurons 27 and in retinal ganglion cells 22'24 have all been shown to be blocked by N B T but not BGT. H o w e v e r , N B T block

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251

Fig. 3. Labeling of DRG neurons with the fluorescent conjugate of a-bungarotoxin with (B) or without (A) pretreatment of the unconjugated toxin. The DRG neuron is brightly labeled by the fluorescent probe; in contrast, the non-neuronal cells are not labeled and thus not fluorescent (A). The fluorescent labeling can be largely blocked by pretreating the culture with 2.5 /~M unconjugated a-bungarotoxin (B), suggesting that binding of the toxin is specific. Final magnification is 550x.

of nicotinic responses was found to be reversible in rat sympathetic neurons 27, but recovery was very slow and incomplete in retinal ganglion cells from rat 1"21. Nicotinic AChRs studied in acutely isolated neurons from medial habenular nucleus were found to be insensitive to both B G T and NBT a°. Along these lines, we investigated the effect of both toxins on the nicotinic responses in D R G cells. Superfusion of the cells with NBT (100 nM) led within minutes to a progressive and nearly complete block of DMPP induced inward currents in the D R G cells. The NBT block of the nicotinic responses was largely irreversible for at least 75 min, the longest time measured by recording from single D R G cells. B G T (1/~M), however, did not significantly affect the nicotinic responses in D R G cells. Nicotinic agonistinduced currents were largely unaffected even when the toxin was present in the superfusion medium for more than 10 min. Effects of both toxins on DMPP-induced inward currents in a single D R G cell are shown in Fig. 2.'

Fluorescence labeling with a-bungarotoxin Specific binding sites for a-bungarotoxin have been found on sensory neurons 2s'3~. As shown above, however, a-bungarotoxin had no effect on the electrophysiologically recorded nicotinic responses. In order to demonstrate specific a-bungarotoxin binding despite the toxin's ineffectiveness as a blocker of the nicotinic responses, we used fluorescent conjugates of a-bungarotoxin to label D R G cells. Freshly dissociated D R G neurons (from the same batches as in our electrophysiological recordings) were brightly labeled by fluorescent a-bungarotoxin in contrast to the non-neuronal cells (probably fibroblasts and glial cells), indicating that nonspecific binding of the fluorescent probe was insignificant (Fig. 3A). Following pretreatment with 2.5 btM unconjugated a-bungarotoxin (from the same lot used in the electrophysiological studies), the number of fluorescent D R G neurons, as well as the brightness of fluorescence of the labeled cells, were significantly reduced although not completely eliminated (Fig. 3B). Additional experiments were performed in the presence of acetylcholine or nicotine. Acetylcholine (200/~M) or nicotine (100/~M) did not appear to block labeling of the D R G neurons by fluorescent a-bungarotoxin (not illustrated). DISCUSSION Our results show that freshly dissociated D R G neurons from 2-day- to 1-month-old rats possess functional nicotinic acetylcholine receptors. These receptors are blocked by neuronal bungarotoxin but not a-bungarotoxin, in contrast to the reverse action of these toxins on nAChRs from skeletal muscle. Thus, nAChRs in rat sensory neurons can be classified as neuronal nAChRs. The electrophysiological and pharmacological properties of these receptors are somewhat similar to those described for nAChRs in rat retinal ganglion cells 21, sympathetic neurons 27 and chick ciliary ganglion neurons 26. Their high sensitivity to blockage of channel function by NBT, however, dearly distinguishes these receptors from those described recently in acutely isolated neurons from the medial habenular nucleus of the rat 30.

Comparison with nAChRs on retinal and autonomic ganglion cells Sensitivity to block of receptor function by NBT seemed to be higher in D R G and retinal ganglion cells when compared to sympathetic neurons. This is evident in the long duration and incomplete reversibility of NBT-induced block in the former 1'21 compared to the faster reversibility in the latter cells 6'27. Higher sensitivity to NBT was attributed to the aa-subunit compared to the

252 a4-subunit in recombination experiments in oocytes 25, but recent findings suggest that the fl-subunit can also influence sensitivity9. However, the exact stoichiometry of the subunits in these nicotinic receptors is not yet known. Both a 3- and ct4- together with flE-SUbunit m R N A have been localized by in situ hybridization in the ganglion cell layer of the rat retina ~6. It is thus conceivable that nicotinic receptors on both rat retinal ganglion and D R G cells are predominantly of the aa-type (high sensitivity to NBT), while the nicotinic receptors on rat sympathetic neurons may be composed mainly of the a4-subunit (lower sensitivity to NBT). It is possible that the apparent partial reversibility of the NBT blockade in both retinal ganglion cells 1'21 and in D R G cells is indeed due to the presence of receptor heterogeneity in these cell types. In fact, a nicotinic receptor insensitive to both BGT and NBT was recently described in acutely isolated neurons from the medial habenular nucleus of rat a°. The amplitude of the nAChR-induced whole-cell currents in freshly dissociated D R G cells was larger compared to retinal ganglion cells from postnatal rat 2x but smaller than in neurons acutely isolated from the medial habenular nucleus of 10-20-day-old rats 3° or cultured embryonic ciliary ganglion neurons from chick z6. Average cell diameter of the D R G cells, however, is up to 1.5-2 times the size of chick ciliary neurons 26, medial habenular nucleus neurons 3° and retinal ganglion cells 1' 2t. Nicotinic responses have been found in approximately 50% of freshly dissociated chick D R G cellsa and the maximal current amplitude was 30% or less compared to the nicotinic responses in chick ciliary ganglion neurons 26 in accordance with our findings in rat D R G cells. An estimation of the number and density of functional nAChRs in single cells may be obtained by calculation based on single channel parameters. Single channel conductance measurements have been performed in retinal ganglion 21, medial habenular nucleus a° and sympathetic neurons from rat 26 and ciliary ganglion cells from chick 26. Moreover, rat neuronal nAChRs have been expressed in Xenopus oocytes after injection of in vitro synthesized RNA and their single-channel currents have been measured aa. The single channel conductances measured in these studies varied between about 13 pS for oocytes injected with a4f12 m R N A and 47 pS in retinal ganglion cells. The single channel conductances determined in sympathetic neurons 27 and retinal ganglion ceils21 from rat as well as chick ciliary ganglion cells e6 were all very similar with 37, 47 and 40 pS, respectively. Along these lines, assuming a single channel conductance of 40 pS with an opening probability of 0.5, freshly dissociated D R G cells from newborn animals would have an average density of only 0.0125 functional nAChRs per pm 2. In an average D R G cell far less than 100 functional

receptors per soma would account for the typical wholecell response compared to about 2000 functional nAChRs in chick ciliary ganglion neurons 26. Thus with only 50% of all D R G cells responding and a very low channel density per cell, the probability of obtaining recordings of single nicotinic channels in excised patches from rat D R G cells is very low and in fact has so far not been achieved in our laboratory. Do nAChRs on D R G cells have a function in vivo? Nicotinic responses were found in freshly dissociated D R G cells from both newborn and 1-month-old animals suggesting that these receptors are not a mere remnant of earlier developmental stages but serve a specific function. This suggestion is underlined by the observed age-related increase in the density of the n A C h R mediated current. Moreover, this finding points to the presence of mechanisms regulating nAChR function in rat D R G cells34. Peripheral endings of sensory fibers have been shown to be sensitive to ACh 32. A 'routing accident' via axonal transport of AChRs involved in dendritic synaptic transmission has been proposed as explanation for the cholinergic responses in sensory endings 35. Our findings clearly show that cholinergic receptors are present on the cell body of D R G cells, at least in vitro. The failure to detect nicotinic responses on the cell body of D R G cells in rabbit 17 reported previously may be explained by receptor desensitization due to prolonged administration of the drug. Desensitization has been found in nAChRs independent of differences in their pharmacologic properties21,26,27,30. D R G cells relay sensory information from the periphery to the central nervous system and are presynaptic to dorsal horn neurons in the spinal cord. Nicotinic receptors have been implicated in the presynaptic modulation of transmitter release in the nigrostriatal dopamine system 7"12. It is conceivable that nAChRs in D R G cells could subserve a comparable function in the regulation of neurotransmitter release in the dorsal horn as presynaptic elements of a feedback loop. However, neurotransmitter receptors found on the soma of freshly dissociated D R G cells in vitro might represent extrasynaptic rather than or in addition to presynaptic receptors. Recently, the possible importance of neurotransmitters in the regulation of neuronal outgrowth, plasticity and survival has been discovered 19. Nicotinic AChRs have been implicated in the modulation of neurite outgrowth in retinal ganglion cells from postnatal rat z2 and stimulation of nAChRs in these cells has been shown to lead to retraction of neurites 5. It may be speculated along these lines that nAChRs could play a role in automodulation of neurite outgrowth in D R G cells. Developmental modulation of D R G cells by both neural

253 and n o n - n e u r a l cells has b e e n o b s e r v e d in cell culture experiments11,29.

p r e s s i o n of n A C h R s

in d i f f e r e n t parts o f the n e r v o u s

system.

CONCLUSIONS Freshly

dissociated

DRG

cells f r o m

postnatal

rat

express f u n c t i o n a l n A C h R s o f a n e u r o n a l type. N i c o t i n i c a g o n i s t - i n d u c e d w h o l e - c e l l c u r r e n t s a p p e a r to i n c r e a s e w i t h age. C o m p a r i s o n retinal

ganglion,

with nicotinic r e s p o n s e s in rat

sympathetic,

and

medial

habenular

n e u r o n s p o i n t s to f u n c t i o n a l diversity and specific ex-

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Acknowledgements. We would like to thank Dr. Elias Aizenman for helpful discussions, and Jeffrey T. Offermann for expert technical assistance. This work was supported by NIH Grants NS-00879 and EY-05477 (to S.A.L.), by a grant from the Sunny von Bulow Coma and Head Trauma Research Foundation (to S.A.L.) and by The Children's Hospital Mental Retardation Core Grant HD-06276. T.P.O.C. was supported by NIH Training Grant NS07264. S.A.L. is an Established Investigator of the American Heart Association.

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