Serotonergic excitation from dorsal raphe stimulation recorded intracellulary from rat caudate-putamen

Brain Research, 243 (1982) 49-58 Elsevier Biomedical Press

49

Serotonergic Excitation from Dorsal Raphe Stimulation Recorded Intracellularly from Rat Caudate-Putamen M. R. PARK, J. A. GONZALES-VEGAS* and S. T. KITAI Department of Anatomy, Michigan State University, East Lansing, MI (U.S.A.) (Accepted December 3rd, 1981) Key words: serotonin - - caudate-putamen - - intracellular recording - - dorsal raphe

Similar to other afferents to rat caudate-putamen, stimulation of the dorsal raphe nucleus evokes a series of 3 responses which can be recorded intracellularly. An initial depolarization is followed by a long-lasting inhibition which is, in turn, terminated by another period of depolarization. Pharmacological manipulations demonstrate that the initial depolarizing potential is serotonergic. Depletion of serotonin by means of prior treatment with para-chlorophenylalanine leads to a reduction in the amplitude of the depolarization which can be evoked by maximal stimulation of dorsal raphe. Neither the long-lastinghyperpolarization nor the late excitation which follow the initial depolarization is affected. Replacement of serotonin levels by injection of 5-hydroxytryptophan results in a restoration of the amplitude of the depolarizing response. The latency of the initial depolarization is, however, unchanged in serotonin-depleted animals. This together with the observation in some cells of a component of the initial depolarization resistant to para-chlorophenylalanine treatment, suggests that there is a non-serotonergic excitation which precedes that mediated by serotonin. INTRODUCTION Confirmation of a projection from the dorsal raphe (DR) nucleus to the caudate-putamen (CP) had to await the development o f tracer techniques which take advantage of axonal transport mechanisms. Data from retrograde transport of horseradish peroxidase (HRP)19,24, zS, anterograde transport of labeled amino acids4,S, 11, and recently, retrograde transport of fluorescent tracers 3~ as well as immunohistological techniques 30 all provide anatomical evidence for this projection. Several lines of evidence indicate that this is a serotonergic projection. Cell bodies in D R labeled after striatal injection of HRp19, 24,25 correspond in location and size to serotonion-containing neurons as determined by earlier histofluorescence studies 12. Histofluorescence techniques have allowed visualization of fine serotonergic axons coursing through CP 13. Striatal 5-HT content is known to decrease upon lesioning of D R 2.

Electron microscopic studies report serotonergic presynaptic terminals in CP as demonstrated by uptake of tritiated 5-HT1,3,10,14. The functional nature of the raphe inputs to CP has not been fully elucidated in that experiments designed to determine whether the raphe-striatal projection is excitatory or inhibitory have produced conflicting results. Nearly all of these have employed extracellular single-unit recording and have reported a depression of activity following stimulation of D R 26 or upon the iontophoretic application of 5-HT in the CP 15,38, although there is one iontophoretic study reporting the opposite result e. When intracellular recordings are made, it becomes clear that the initial transmembrane event occurring as a result of D R stimulation is a depolarization a2. The depolarization can be subthreshold for the generation of action potentials and therefore be undetectable to an extracellular microelectrode. This excitatory potential has been reported to be monosynaptic and to

* On leave from: Escuela de Bioanalisis, Facultad de Medicina, Universidad Central, Caracas, Venezuela.

50 have a latency of 4-8 ms (mean 4.9 ms) and a duration of 15-20 ms 32. Only after it ceases can an inhibitory potential, which is of remarkably long duration (150-300 ms) be observed in striatal neurons. In evaluating these results, the initial EPSP has been identified as the monosynaptic dorsal raphe response, primarily based on the arguement that the subsequent events (long-lasting inhibition and rebound excitation) are not specific to DR stimulation but are seen following stimulation of any of the other afferents to CP 9,16,17,32. These latter are cerebral cortex, intralaminar thalamus, and substantia nigra. The experiments reported herein are designed to be a direct test of the proposal that the initial EPSP is the serotonergic response. The approach chosen is to compare the intracellular postsynaptic responses resulting from DR stimulation under conditions in which the amount of 5-HT available for synaptic transmission is changed. The drugs used to alter 5HT levels are para-chlorophenylalanine (PCPA), a specific blocker of the synthetic enzyme tryptophan hydroxylase and 5-hydroxytryptophan (5-HTP), the immediate precursor of 5-HT ~°. When administered intraperitoneally, PCPA depletes 5-HT levels 90 within 24 h 20. Restoration of 5-HT follows the administration of 5-hydroxytryptophan (5-HTP) with a time-course which is rapid enough to be observed during the acute recording session20, ~5,~6. In the present study, manipulation of the amount of 5HT available as a neurotransmitter results in corresponding changes in the amplitude of the initial EPSP but not the subsequent events. MATERIALS AND METHODS

15-20 s during a trial period in which the respirator was turned off. The animals were then paralyzed with D-tubocurare (0.1 mg/kg, i.v.). lntracellular recordings were performed using glass microelectrodes pulled on a Narashige vertical puller from 2 or 3 mm diameter omega-dot capillary glass (WPI Instruments). Microelectrode tip diameter was judged smaller than 0.2 #m as it was beyond resolution when viewed through a light microscope equipped with 100 × dry metallurgical objectives designed for use without a coverglass (Olympus MPlan 100). The electrode was filled with either 3 M KC1 or 2 M potassium methylsulphate (ICN Pharmaceuticals). Standard intracellular recording techniques were used. The biological amplifier employed was a Bioelectric PI equipped with an active bridge for current injection. Stimulation of the dorsal raphe nucleus was monopolar using a stainless-steel insect pin insulated to within 0.5 mm of the tip. The DR stimulating electrode was stereotaxically placed at the rostral end of the nucleus, in the midline, at coordinates 1.2 mm anterior and --1.6 vertical, using the system of K6nig and Klippe121. This electrode placement has been found optimal for activating ascending raphe projections to striatum 3a. Bipolar stimulating electrodes were placed in cerebral cortex and substantia nigra as has been previously described 2s. Stimulation consisted of single electrical pulses 0.05 ms in duration and 5-50 V in amplitude. Intracellular recordings were made in the head of the caudate-putamen. After the recording session, animals were perfused with 10 % formalin, sectioned and stained with neutral red for histological verification of stimulating and recording sites.

Recording techniques Experiments were performed on male LongEvans rats whose weights ranged from 280 to 330 g. The animals were anesthetized with an intraperitoneal injection of urethane (1.0 g/kg). A cannula was placed in the femoral vein for the i.v. injection of drugs. A tracheal cannula was inserted and the animals artificially ventilated with 100~o 02 at a respiratory rate of 70 strokes/min. The animals were maintained slightly hyperkapnic by adjusting the tidal volume at the beginning of the experiment so that the unparalyzed animal remained apneic for

Drug administration In experiments where 5-HT levels were to be manipulated, PCPA was administered beginning 3 days prior to the recording session. An initial intraperitoneal dose of 375 mg/kg was given at this time. This treatment, at a nearly identical dose, has been shown to reduce 5-HT levels by 9 0 ~ 2° and to produce detectable effects on central serotonergic neurotransmission35, 36. A supplemental injection of 100 mg/kg was given 24 h before recording, also i.p. To acutely restore 5-HT levels, 5-HTP was admi-

51 nistered intravenously during intracellular recording at a dose o f 15 mg/kg. The desired o u t c o m e o f the P C P A / 5 - H T P experiments is a time series o f responses internally controlled and dependent u p o n the maniptdation o f 5H T level. This requires a continuous and stable intracellular recording for the entire time o f 5 - H T P effect. As approximately 15 min passed before an

A

effect o f 5 - H T P was seen, and sufficient time before and after that period is needed to gather data, the required m i n i m u m duration o f stable intracellular recording is 45 rain. Experiments in which the complete set o f internal controls were n o t obtained could still provide corroborative data by comparing the responses o f different cells encountered before and after the 5 - H T P injection.

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Fig. 1. Intracellular responses to stimulation of dorsal raphe, cerebral cortex, and thalamus. Each record consists of 4 traces: low gain DC intracellular trace, high gain AC-coupled trace, below which are high gain AC extracellular control and low gain DC extracellular control. In A, D, and F the intracellular traces are made up of two or more superimposed oscilloscope sweeps. Calibration bar for high gain AC represents 4 mV and for low gain DC, 40 inV. A-C: the set of transmembrane events which result from stimulation of dorsal raphe. An initial depolarization is followed by a long-lasting hyperpolarization which is itself terminated by a period of depolarizing potentials. Similar responses occur following stimulation of cerebral cortex (D, E) and intralaminar nuclei of the thalamus (F, G).

52 RESULTS

Effect of PCPA treatment

Normal response The response o f CP neurons to stimulation of D R is a sequence o f events consisting of: (1) an initial depolarization; followed by (2) a hyperpolarization with a duration in the order o f 200 ms; which is in turn terminated by (3) a 100-300 ms period o f increased excitatory drive (Fig. 1A - C ) . This series o f responses is similar to those observed following stimulation o f cerebral cortex (Fig. 1D, E), and thalamus (Fig. IF, G). The initial EPSP evoked by D R stimulation can reach threshold and trigger action potentials, as is illustrated in Fig. 1B and C.

A

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O f these various responses, only the early depolarization resulting from D R stimulation is sensitive to manipulations o f 5-HT levels. Thus, animals treated with P C P A prior to the recording session typically show no initial depolarization or one o f only low amplitude (less than 2 mV) as a result o f stimulating D R (Fig. 2 A - D ) . The response in Fig. 2A and B is 1.0 mV in amplitude, which compares with a 7 mV E P S P in the same cell resulting from stimulation o f substantia nigra (Fig. 2E-F). N o r mally, responses to D R are comparable in magnitude to those elicited by stimulation o f substantia nigra, with similar stimulus currents. Regardless of

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EPSP Amptifude Fig. 2. Responses in PCPA-treated animals. A-D: the responses to DR stimulation recorded from 2 neurons in 2 different animals pretreated with PCPA. E-G: the corresponding control responses in the same two neurons to stimulation of substantia nigra. A, B and E, F are from the one neuron and C, D and G are from the other. Each record consists of high gain AC traces recorded intracellulariy (above) and extracellularly (below). Records C-G also depict the low gain DC traces. PCPA sharply reduces the intial depolarizing response to DR stimulation (A-D) whilst the late responses and the early depolarizing response to substantia nigra stimulation remain large. H-I: frequency histogram of the maximal depolarizing responses to DR stimulation in PCPAtreated animals (I) and untreated animals (H).

53

Effect of 5-HTP replacement

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Latency (msec) Fig. 3. Frequency histogram of the responses to dorsal raphe stimulation in normal and PCPA-treated animals. No change in latency is apparent.

which afferent is stimulated to evoke them, the longlasting hyperpolarization as well as the late depolarization are still present in PCPA-treated animals. Examples of these PCPA-resistant late responses are illustrated in Fig. 2, both following stimulation of D R (Fig. 2B, D) as well as SN (Fig. 2F, G). Frequency histograms of the maximal amplitudes of the initial depolarization resulting from D R stimulation in control and PCPA-treated animals are portrayed in Fig. 2H and I. In control animals the mean response for 22 neurons was 3.3 mV. Only one neuron was encountered which had a response of less than 0.5 mV. In contrast, 21 of 58 neurons recorded in PCPA-treated animals had responses less than 0.5 mV in amplitude. The mean response was 1.3 inV. Pretreatment with PCPA had no affect on the distribution of latencies of the initial depolarizing response. Fig. 3 allows comparison of the latencies of non-treated (Fig. 3, mean ---- 7.2, n ---21) with those of PCPA-treated ones (Fig. 3, mean = 6.7 ms, n = 36). The lower histogram of Fig. 3 contains only data from neurons in which the initial response was not completely abolished and therefore from which latency could not be measured. These two latency distributions cannot be shown to differ significantly (t = 0.60, df -- 55, P > 0.25).

5-HTP was injected intravenously during the course of recording from animals previously treated with PCPA in order to observe the result of restoring 5-HT levels. For the majority of experiments, it was only possible to observe the restoration of an excitatory response with 5-HTP by comparing the amplitudes of maximal responses in successive intracellularly recorded neurons. This is illustrated in Fig. 4 for a series of 24 neurons recorded in a single animal. The first 15 cells encountered in this PCPAtreated animal in no case showed a response to maximal D R stimulation larger than 3 mV. The vast majority (12 of 15) have a maximal response of less than 2 mV. Following the injection of 5-HTP, there was a large transient increase in the amplitude of the D R response. The initial response was 11.5 mV in amplitude 30 min after 5-HTP injection (cell 16, Fig. 3). The next cell encountered and all subsequent cells had maximal responses of less than 5 mV, indicating recovery from the action of 5-HTP. Cell 17 was recorded 60 min after the 5-HTP injection. In 3 cases it was possible to observe the D R evoked response increase in amplitude, in a single neuron, as a result of 5-HTP injection. Fig. 5 illustrates one of these cells. In the 5-HT depleted state, 30 min after the administration of 5-HTP, the amplitude of the maximal D R response was 1.5 mV. In the course of the next 30 min, the response increased in amplitude to 5 mV. During this time the

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Fig. 4. Histogram plot of the maximal EPSP amplitude resulting from D R stimulation in 24 neurons successively recorded from a single PCPA-treated animal. Between cell 15 and 16, an i.v. injection of 5-HTP (15 mg/kg) was made. The next cell encountered (cell 16) exhibited a maximal EPSP amplitude much larger than the preceding cells. The amplitude of subsequent intracellularly recorded neurons approaches that of the pre-5-HTP group.

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Fig. 5. Growth of an intracellularly recorded excitatory response to DR stimulation as a result of 5-/-ITP injection. The amplitude of the EPSP increases from 1.5 mV (A) to 5 mV (C) and is able to trigger an action potential. D is the extracellular control• The amplitude of the EPSPs recorded in this neuron are plotted in E as a function of the time after 5HTP injection• For the time until the maximal effect was reached, resting membrane potential was stable. The cell then began to hyperpolarize, during the period in which the response was recovering.

resting m e m b r a n e p o t e n t i a l r e m a i n e d relatively constant at - - 5 0 mV. In the later phases o f the recording, the cell b e c a m e hyperpolarized. In spite o f this increase in resting m e m b r a n e potential, which t a k e n alone w o u l d have the effect o f increasing the a m p l i tude o f a n y E P S P m e d i a t e d b y a c o n d u c t a n c e increase, the a m p l i t u d e o f the D R response decreased, p r e s u m a b l y m a r k i n g the recovery f r o m the effects o f 5-HTP. The records o f Fig. 5 A - D show traces o f the p o t e n t i a t i n g D R response. The latency o f the c o m p o n e n t p o t e n t i a t i n g after 5 - H T injection is 14 ms. L a t e n c y o f this latter c o m p o n e n t is m e a s u r e d from the time o f s t i m u l a t i o n to the p o i n t where the 5 - H T P p o t e n t i a t e d response first deviates f r o m the traject o r y o f the p r e - 5 - H T P response• W h i l e the D R response increases as a result o f 5H T P injection, the E P S P e v o k e d f r o m stimulation o f a n o t h e r afferent does n o t (Fig. 6). F o l l o w i n g a d m i n i s t r a t i o n o f 5-HTP, which occurred 5 min before trace A in Fig. 6, the D R responses increase in a m p l i t u d e f r o m 8.2 mV to 20 mV. D u r i n g this same p e r i o d the E P S P e v o k e d f r o m s t i m u l a t i o n o f s u b s t a n t i a nigra m a i n t a i n s a c o n s t a n t a m p l i t u d e (Fig. 6 E - H ) . In P C P A - t r e a t e d animals there were always a few

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Fig. 6. Growth of the depolarizing response to DR stimulation following injection of 5-HTP. A-D: there is an increase in amplitude of the DR-evoked response from 8.2 mV to 20 mV (D) as a result of 5-HTP injection. E-H: the response to substantia nigra stimulation recorded at corresponding times remains constant. Extracelhilar controls are indicated by the broken lines• responses which were relatively large, up to 6 mV. In a t t e m p t i n g to differentiate these f r o m serotonergic responses, it was f o u n d t h a t the P C P A resistant responses were n o t r e d u c e d in a m p l i t u d e in d o u b l e shock experiments (Fig. 7A, B). The d o u b l e - s h o c k p r o c e d u r e consists o f p a i r i n g the test stimulation to D R with a preceding c o n d i t i o n i n g stimulus, to either D R or to a n o t h e r afferent• The c o n d i t i o n i n g stimulus in the case illustrated in Fig. 7

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Fig. 7. Paired stimulation distinguishes PCPA-resistant and 5HTP potentiated responses. A and B are, respectively, the responses in a PCPA-treated animal to a single test stimulus (A) and to paired stimuli (B). Paired stimuli consisted of the test stimulus preceded by a conditioning stimulus to substantia nigra. Interstimulus interval is 50 ms. The test (second) response is not reduced in amplitude by the preceding conditioning stimulus• C, D : single and conditioned stimuli for a DR response which had grown to over 7 mV in a PCPAtreated animal following 5-HTP injection. In contrast to the PCPA-resistant response (A, B), this response is sensitive to 5HTP manipulation, and is virtually abolished when preceded by the conditioning substantia nigra stimulus.

55 B, D is to substantia nigra. This lack of reduction in amplitude contrasts with the behavior of the EPSPs which can be recorded in the same population of animals (PCPA-treated) but whose amplitude has been resorted after injection of 5-HTP. It is characteristic of these post-5-HTP responses that the test response is completely abolished following a conditioning stimulus (Fig. 7C, D). DISCUSSION The present results confirm the findings of VanderMaelen et a133 in showing that the initial response which occurs as a result of stimulation of the dorsal raphe is a depolarization and that this is followed by a sequence of 2 other intracellular events, a long-lasting hyperpolarization, in turn terminated by a period of depolarization. The purpose of the present study is to determine which of these intracellularly recorded responses is due to serotonergic neurotransmission. The method of choice for the study of central serotonergic transmission is to employ PCPA, the well-known depleter of 5-HT, to experimentally lower the content of 5-HT in presynaptic terminals. PCPA is a specific blocker of tryptophan hydroxylase, the rate-limiting step in the synthesis of 5-HT 2°. When administered as has been done in the present study, PCPA reduces the 5-HT content assayed in whole brain by more than 90 ~20. The complement to PCPA in the experimental paradigm is 5-HTP, which serves to restore 5-HT levels20,35 during the recording session. 5-HTP is able to cross the bloodbrain barrier and is the substrate for the decarboxylation reaction which is the final step in the synthesis of 5-HT 2°. The results of the pharmacological manipulations performed in this study indicate that the initial depolarization and it alone constitutes the serotonergic response. Of the 3 elements of the response to DR stimulation, only this initial depolarization is affected by depletion of 5-HT. Acute administration of 5-HTP leads to restoration of the amplitude of the EPSP. Moreover, the time-course of restoration of the 5-HT response corresponds well to the time course of restoration of 5-HT content of whole brain in PCPA-treated animals2o. These authors found a sharp increase in 5-HT content within 30 min of a

subcutaneous injection of 5-HTP. Other studies have noted recovery of 5-HT dependent physiological~5 or behavioral (e.g. ref. 5) responses within 30 min. The other parameters not related to the restoration of serotonergic neurotransmission which could account for an increase in amplitude of an intracellularly recorded EPSP have been controlled for. These parameters would be an increase in either resting membrane potential or input resistance of the recorded neuron. We have demonstrated that the DR response increases without a concomitant change in membrane potential (Fig. 5). Additionally, in comparing the waxing DR response with an unchanging response from another set of afferents (substantia nigra stimulation, Fig. 6) we have shown that changes in both membrane potential and input resistance which could affect the amplitude of a postsynaptic response are not occurring. The initial depolarization, however, does not appear to be purely serotonergic. Several sets of observations suggest that it consists of two components, both excitatory. Thus, while PCPA treatment dramatically reduces the maximal amplitude of DR evoked responses, depolarization of over 0.5 mV can still be recorded in slightly over half the neurons encountered. In some cases the magnitude of the PCPA-resistant response is quite large (Fig. 7A). It is unlikely that these remaining responses are due to incomplete depletion of 5-HT as they behave differently from demonstrably serotonergic responses in double-shock experiments. That is, following injection of 5-HTP, the response which increases in amplitude, and therefore is serotonergic, is abolished by a preceding conditioning shock whereas the PCPA-resistant response is not (Fig. 7). The distribution of latencies of the DR-evoked response (Fig. 3) suggests on two counts that the non-serotonergic component is carried by faster conducting fibers than the serotonergic one: (1)the latencies observed are short; and (2) the distribution is unchanged in PCPA-treated animals. The best estimate of what the latency of serotonin-mediated responses in CP should be comes from conduction velocity measurements of rostrally-projecting dorsal raphe neurons. These have been measured to lie in the range of 0.5-1.5 m/s, with the bulk being less than 1.0 m/s z4. Recent intracellular data from DR

56 neurons confirm these values 27. At this conduction velocity, the latencies for monosynaptic 5-HTmediated events recorded in CP should be no less than 5 ms, and most should be greater than 7 to 8 ms. This is based on a straight-line conduction distance of 7.5 mm from stimulating to recording site. The true course of rostrally projecting dorsal raphe axons is longer than the straight-line distance4, is so that this estimate of latency is biased toward low values. The bulk of the responses observed in this study fall in the range 2-10 ms, with means of 6.7 and 7.2 ms for control and PCPA-treated animals, respectively. These values are similar to those obtained by VanderMaelen et al., who report a range of 4-8 ms. However, the briefest latencies measured in either the present study or that of VanderMaelen et al. 33 would correspond to conduction velocities faster than any reported for efferent fibers of the dorsal raphe nucleus. The mean and modal latencies of the observed responses only just reach the range predicted for serotonergic axons. The identity of the non-serotonergic component which accounts for the short latencies is not known, although preliminary data indicate that it is of non-raphe origin (M.R. Park, unpublished observations). In the neostriatum, the bulk of opinion has favored an inhibitory role for serotonin. The data which have contributed to this view are from extracellular single unit recordings in which dorsal raphe is electrically stimulated 26 or in which 5-HT is iontophoretically applied 3s. Intracellular studies, however, both the present one and that of VanderMaelen et al. 33, indicate that 5-HT is an excitatory neurotransmitter in caudate-putamen. This requires a reappraisal of the extracellular results: In their extracellular single-unit study, Olpe and Koella 26 concluded that the long-lasting inhibition following D R stimulation is serotonergic. This was based on experiments where the inhibition could be antagonized, in 9 of 15 cells, after protracted (2-4 min) iontophoretic administration of methysergide. Sensitivity to methysergide, however, is not a satisfactory test for serotonergic transmission in the central nervous system in that various reports ~5,~6 have shown its lack of antagonism to central serotonergic transmission. Our results indicate that neither the long-lasting hyperpolarization nor the excitatory surge which generally follows it can be causally

linked to activation of serotonergic afferents to caudate-putamen. Were the prolonged hyperpolarization a monosynaptic serotonergic IPSP or a polysynaptic response with a serotonergic link, it would have been abolished in PCPA-treated animals. Moreover, our data confirm previous observations 9, 16,17,33,37 that the long-lasting hyperpolarization is elicited following stimulation o f any of the striatal afferent systems (i.e. cerebral cortex, substantia nigra, or thalamus). It is now known that intrinsic inhibition in striatum cannot account for the prolonged inhibition 22,28. Recent intracellular data show the long-lasting hyperpolarization to be dependent upon neuronal elements located in cerebral cortex 37. The presence of the late and longlasting inhibition in 5-HT-depleted animals strongly argues against the existence of a 5-HT-mediated inhibition in neostriatum. The conclusions ofiontophoretic studies that 5-HT is an inhibitory neurotransmitter 15,8s (although see Bevan et al. 6 for the opposite interpretation) are not so easily accounted for with the present data. There are, however, sufficient differences in the two approaches, iontophoretic application of an agonist as opposed to electrical stimulation of an afferent, that opposite results need not be interpreted as contradictory. Thus, the effects resulting from the iontophoretic application of an agonist may involve extrasynaptic receptors whose action differs from that of the subsynaptic membrane 7. In this regard, there is the recent observation that 5-HT iontophoretic responses can be antagonized by the GABA antagonists bicuculline and picrotoxin 2z. This could be a consequence of a nonspecific action of both bicuculline and picrotoxin (unlikely as they act at different sites zg) or a 5-HT action on GABA receptors. In this study, we have relied upon intraccllular recording techniques. This has allowed the resolution of excitatory responses which are often subthreshold for the generation of action potentials and which, therefore, cannot be detected by the extracellular single unit electrode. This would secm to be the principle reason that the excitatory action of serotonin in caudate-putamen has not been detected earlier.

57 ACKNOWLEDGEMENTS

T h e authors w o u l d like to t h a n k J a m e s W. Lighthall f o r his assistance in the early experiments o f this

This w o r k was s u p p o r t e d by U S P H S N I H G r a n t N S 14866 to S.T.K. a n d N I H B R S G G r a n t R R

project an d Charles J. W i l s o n f o r his c o m m e n t s on the manuscript.

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