• Abstract

• Introduction

• Materials and Methods

• Results

• Discussion

• Acknowledgments

• References
  

ABSTRACT

Recently, abuse of the hallucinogenic drug methamphetamine (MA), known on the street as "crystal", has increased. It has not been conclusively determined whether MA causes depletion or degeneration of serotonin (5-HT), as well as dopamine system, in the brain. This important difference is difficult to resolve since detection markers are subject to 5-HT levels. In this study, using immunocytochemistry with antibodies against the serotonin transporter (5-HTT), we report that MA at doses of 25-50 mg/kg i.p. caused dose dependent damage to 5-HT fibers within hours. An apparent varicosis of 5-HTT immunoreactive (im) fibers is reported for the first time, which has not previously been seen using anti-5-HT antibodies. At 25-50mg/kg, the vesicular cysts enlarged from 2 to as large as 15 µm, and began to perforate when exceeding approximately 7 µm. At 50mg/kg, in addition to the vesicular enlargement, a complete disintegration and disappearance of a small population of 5-HTT-im fibers in the subareas of the cortices occurred. A subpopulation of 5-HTT-im fibers had already been reduced in density within hours after injection. Four days after MA injection, perforated 5-HTT-im fibers became fragmented, which may have led to the degradation of distal 5-HT fibers. The 5-HTT-im fibers were greatly reduced in many regions of the brain, particularly in the frontal and parietal cortices, hippocampus, striatum, and thalamus. In summary, staining with our 5-HTT antibody confirmed that 25-50mg/kg MA causes structural damage and axonal degeneration of 5-HT fibers. In addition, for the first time, we report that depending on the dosage, there are two phases of 5-HT fiber damage caused by MA: (a) acute phase: varicosis, perforation, and disintegration of 5-HTT-im fibers within hours, and (b) chronic phase: fragmentation and appearance of terminal stumps over days. The definition of 5-HT fiber degeneration is discussed. As a result of 5-HT fiber destruction, MA administration would most likely alter the normal 5-HT function in the brain for an extended period of time.

INTRODUCTION

Degeneration versus depletion

A number of hallucinogens and psychostimulants, including LSD, cocaine, 3,4-methylenedioxymethamphetamine (MDMA, Ecstasy) , 3,4-methylenedioxyamphetamine (MDA), methamphetamine (MA, Crystal) and various other analogs of amphetamine, are known to have major effects on serotonin (5-HT) [and/or dopamine] neurons. These chemicals, through different routes in different species, alter or in many cases decrease 5-HT and/or 5-HIAA in areas of the brain (De Souza et al., 1990; Gibb et al., 1987; 1990; De Souza and Battaglia, 1989; Sanders-Bush et al., 1988; Kleven & Seiden, 1989; Sonsalla et al., 1989; Sonsalla & Heikkila, 1988; Ricaurte et al., 1980, 1985,1988; Finnegan et al., 1988; Schmidt & Taylor, 1988; Pierce & Peroutka, 1990; Woolverton et al., 1989; Stone et al., 1986). This observation leads to an important question: "Are serotonergic neurons degenerated by these compounds ?" Immunocytochemical staining, using 5-HT antibody, shows that a large number of 5-HT immunostaining fibers disappear from the brain (Axt & Molliver, 1991; Appel et al., 1989; O'Hearn et al., 1988). In some cases, the reduction in immunostaining fibers seems to be long-lasting (MA and MDMA); in other cases, the fibers reappear in varying degrees and at different time periods.

Because the immunocytochemical visualization of 5-HT neurons relies on antibodies to 5-HT or to its rate-limiting enzyme, tryptophan hydroxylase (TPH), the anatomical identification of neurons and especially their terminals are dependent upon the antigen level present. When depletion of 5-HT occurs, the population of 5-HT-immunoreactive fibers and cell bodies does not necessarily reflect the true population of the fibers nor the neuronal cell bodies. Whether 5-HT fibers degenerate or the level of 5-HT in the fibers simply becomes too low to be detected by immunocytochemistry, and whether 5-HT fibers regenerate or the 5-HT is replenished to the level of sensitivity of immunocytochemistry, are matters for discussion (Molliver et al., 1990; Sotelo, 1991; Kalia, 1991; Paris & Cunningham, 1991; Zhou et al., 1994). Consequently, the alleged 5-HT fiber degeneration examined by 5-HT/TPH immunocytochemistry is inconclusive.

Realizing that 5-HT immunocytochemical methods are compromised by 5-HT/TPH depletion (Battaglia, 1990), a number of investigators adopted a supplemental analysis of 5-HT degeneration by ligand binding of the uptake site --- biochemically in two laboratories (Battaglia et al., 1987; 1988; Insel et al., 1989; Nash et al., 1991) and autoradiographically in E. De Souza's lab (De Souza et al., 1990; Battaglia et al., 1991). These investigators provide additional evidence of degeneration by detecting missing uptake sites in specific areas of the brain.

Recently we have made three antibodies against the 5-HT transporter (5-HTT) (Zhou et al., in press). 5-HTT satisfies the following criteria as a probe for detecting plasticity of 5-HT fibers: (a) specific to 5-HT neurons, (b) non-diffusible, (c) presynaptic, and (d) capable of revealing morphological details (Zhou et al., in press). The 5-HTT-N antibody was used in the current study.

Abuse of MA, known on the street as "crystal", has lately been on the rise. However, it is one of the least understood drugs in terms of depletive or degenerative effect on 5-HT fibers. The effects of MA on the 5-HT system have been studied immunocytochemically using 5-HT antibodies (Ricaurte et al., 1980; Johnson et al., 1987; Farfel et al., 1992; Richards et al., 1993; Pu and Vorhees, 1995). The definitive effect of MA on damage of 5-HT fibers remains to be determined by a non-5-HT or TPH related markers. Our study has, for the first time, used serotonin transporter (5-HTT) antibody as the marker to examine the 5-HT degeneration, and we report that MA causes definitive damage to 5-HT fibers with peculiar morphological changes.

MATERIALS AND METHODS

1. Animals and Methamphetamine Treatment

Young adult male and female Sprague-Dawley rats (200-250 g) were used for drug testing. The animals were housed individually in the Indiana University Laboratory Animal Research Center under a normal light-dark (0700-0700) cycle with free access to rat chow and water. The room temperature is maintained at 25^oC and humidity at 45%.

MA was administered i.p. using various regimens: 0 (saline vehicle control),12.5 , 25, and 50 mg/kg/day for two different periods of time (see table I). At 2.5 hours or 4 days after the last drug administration, animals were perfused for immunocytochemistry for 5-HTT staining. At the 50 mg/kg/day for 4 days, the highest dose, 3 out of 7 animals died. The descriptions of various dosages on the damage to 5-HTT-im fibers, hereafter, refer to the 4 day-injection period, unless otherwise stated. (Female rats were used for most of the study, except 6 male rats, 2 control and 4 in 50mg/kg/2.5 hr paradigm, were used to detect if gender is a factor. We found MA caused similar damage to central 5-HT system in male as in female rats).

Table I. The animal groups according to dosage and perfusion time.

Dose of MA injection(mg/kg/day)	0	12.5	25	25	50	50
Day(s) of injection	4	4	1	4	1	4
Total amount	0	50	25	100	50	200
Perfusion	4-day	4-day	2.5hr	4-day	2.5hr	4-day
Number of animals	6	5	1	10	6	7

2. Immunocytochemistry of 5-HTT

All animals were perfused intracardially with formaldehyde (Reagent grade, Fisher Scientific) freshly made from 4% paraformaldehyde and 0.1 M phosphate-buffered saline (PBS) under deep Urethane (120 mg/kg) anesthesia. Their brain (regions from 3.0mm anterior and 8.5mm posterior to bregma, which include forebrain [except olfactory bulbs and pituitary gland] and anterior brainstem) were removed, left in the same fixative overnight, and coronally sectioned at 40 µm with a vibrotome for immunocytochemical staining (Zhou et al., 1991, 1994) of 5-HTT. Primary antibodies, antiserum to 5-HT transporter (5-HTT-N, against first 71 amino acids of 5-HTT N-terminal, 1:1000 dilution antiserum, produced and characterized in Zhou's laboratory, Zhou et al., in press) was used. Three sets of controls were tested previously for immunocytochemistry of 5-HTT-N, the preimmune serum of each antiserum, the 5-HTT-N antiserum preabsorbed with fusion protein, and skipping of the primary antiserum. All controls processed under the same conditions in parallel with the 5-HTTN staining showed negative results (Zhou et al., in press). Sheep anti-rabbit was used as secondary antibody. Sternberger's peroxidase-anti-peroxidase (PAP) indirect-enzyme method was used for staining. The PAP reaction was performed with 0.003% H₂0₂ and 0.05% 3'3-diaminobenzidine. The primary, secondary and marker antibodies were diluted with PBS containing 0.3 % Triton X-100 and 1% normal sera of the species from which the second antibodies were raised. The primary antibodies were incubated overnight; the second and third, for one hour each. Three washes with PBS each for 5 minutes were used between each antibody. All the sections were Nissl-counterstained with methyl green to reveal background cells and to profile brain structures.

3. Image analysis of 5-HTT staining

The density of 5-HTT-immunostained (im) fibers/ varicosities in frontal and parietal cortices were compared between control and drug-treated groups, under a dark-field microscope connected to a Macintosh with a NIH image analysis system. Utilizing the immunocytochemistry of 5-HTT-IM fibers, a Leitz Orthoplan II microscope, and a Nevicon 70 camera in our laboratory, we were able to visualize and measure density at the single fiber /varicosity level (Zhou and Azmitia, 1988; Zhou and Buchwald, 1989, Zhou et al., 1991, 1995). The density of 5-HTT-im fibers was examined by Overview-Measurement (measurement of the whole brain nucleus by montage) under 10X objective lens. A box of 0.9 mm x 1.2 mm was chosen in the same region of the frontal and parietal cortices in each section, and were recorded in 6-8 consecutive, but alternating sections. Animals were sacrificed 4 days after either saline, 25mg or 50mg/kg MA injection for fiber density measurements. The relative fiber density (no correction factor was used to convert the area density to volume density) is expressed as % of the background. At least three animals from each group were chosen for comparison of a given region.

Control: Precise consistency in section-thickness and Triton-x penetration is important for density measurements, and was strictly controlled among groups. The immunopenetration done in our laboratory, estimated by Epon-embedded cross-section, is approximately 15µm. In each experiment, control and experimental groups were processed simultaneously under identical conditions. The staining variability among animal was controlled by comparing the referenced brain regions which has most constant staining.

RESULTS

Morphological evidence for dose-dependent damage of the 5-HTT-im fibers was observed in rats treated with MA from 12.5 to 50 mg/kg/day. In 12.5 mg/kg/day dosage, MA caused very minor damage as compared to the saline group in forebrain and brainstem. In the 25 and 50 mg/kg range, a one dose administration caused extensive damage within 2.5hr in frontal, parietal and temporal cortices, hippocampus, thalamus, habenula nuclei, septal nucleus, and major 5-HT pathways, cingulum bundle, fimbria-fornix, indusium griseum, stria terminalis and median forebrain bundle. Degeneration and reduction of 5-HT fiber were seen after 4 days in the above areas. The regimen of 50 mg/kg caused the greatest damage in many brain regions and resulted in the most severe reduction of 5-HTT-im fibers after 4 days in all areas of forebrain, except in brainstem and its raphe.

MA caused characteristic damage to 5-HT fibers depending upon the dosage and time. The degeneration processes are described sequentially below. The first three occurred within hours and are designated as Phase I, the acute phase; the latter two occurred over a period of days and is Phase II, chronic phase.

Phase I.
Acute phase, within 2.5 hours after MA injection.

1. Varicosis

   Within hours of MA administration, 5-HTT-positive fibers had begun to swell and were enlarged at points along their length, and formed large empty varicose or varicoses varying in size from 2-20 µm along the fibers (Fig 1.). Many varicoses were 5-10 µm in diameter, and some extended to 20µm. This major phenomenon was observed for the first time by 5-HTT staining. It occurred across all dosages from 12.5-50mg/kg, but not below our minimum dose (not shown). In 25-50mg/kg doses, the varicosis prevailed in many regions of the brain, including the frontal and parietal cortices, striatum, hippocampus, habenular nuclei, thalamus, septal nucleus, cingulum bundle, indusium griseum, stria terminalis, and medial forebrain bundle.

   Figure 1: Methamphetamine injection caused Varicosis / Perforation and degeneration of 5-HT fibers in the cingulum bundle. The 50 mg/kg MA caused Varicosis, empty vacuous enlargements, in the segment of fibers ranging in size from 2-20µm in diameter (in this section) within hours. A number of varicoses are shown in the septal nucleus (a). The large varicoses (b, arrowheads) were often perforated when observed under the microscope by focusing through the depth of the sections (b, arrows). Four days after MA injection, fragmentation at where varicose perforation occurred (nicked arrowheads), and a typical 5-HT degeneration profile with degradation debris was seen (c,d; crossed arrow). A small number of varicoses were still seen 4 days after injection of MA. Scale bars: a,c,d=20µm. b=10µm.

    

2. Perforation

   Varicose perforation occurred at dosages of 25-50mg/kg. Small varicoses along the fibers seemed intact on their surface, while the large varicoses were either broken or had openings on the surface of the varicose. The large varicoses exceeded approximately 7 µm in diameter and were mostly perforated with an opening size of 2-10µm. The 5-HTT-im fibers, particularly those in the frontal and parietal cortices, fornix, septal nucleus (Fig. 1a,b) and cingulum bundle (Fig. 2c), formed varicoses in the middle of the fiber segments. This process was particularly damaging when varicosis and perforation occurred in fiber bundles such as the cingulum bundle, fornix, and the indusium griseum which sends 5-HT fibers to large cortical and subcortical areas.

   Figure 2: Methamphetamine injection caused Varicosis and degeneration of 5-HTT-im fibers in the cingulum bundle (CB). The 5-HT fibers use CB [and indusium griseum] to reach broad cortical areas (a, low magnification; b, high magnification). Saline control injections showed abundant 5-HTT-im fibers in the CB. MA injections caused Varicosis of 5-HTT-im fibers in the CB (c, stars) within hours. Many fibers later degraded, and only a few remained 4 days after lesion (d). This disruption of the 5-HT path in the CB would affect innervation in the cortical areas. Sept: septal nucleus. Scale bars: a=1.7 mm, b,c,d=20µm

    

3. Disintegration

   Disintegration of 5-HTT-im fibers, the rapid and complete breakdown of 5-HTT-im fibers into an undistinguished form, occurred within a few hours. The disintegration could be attributed to the acceleration of varicosis, perforation and fragmentation, or it may have by-passed the above described processes and reduced into small barely visible debris. It was seen only in the high dose (50 mg/kg/day) MA group, whose 5-HTT-im fiber density already exhibited minor reductions in the frontal, parietal and cingulate cortices, hippocampus, thalamic regions, and cingulum bundle (Fig 2c as compared to 2b) just 2.5 hours after MA injection. This indicates that disintegration of 5-HTT-im fibers can occur within the first two hours. Another feature observed at this stage was rather few or a very small number of terminal stumps. This profile is characteristic of the stereotypical degeneration.

Phase II.
Chronic phase, from 2.5 hours to 4 days after MA injection and beyond. Phase II occurred sequential to Phase I.

1. Fragmentation

   Fragmentation was observed after a few hours to a period of 4 or more days. The 5-HT fibers began to fragment at the varicosis and /or perforated sites (Fig 1c,d). Fragmentation was seen at the same dosages and at the locations where perforation occurred.

2. Degradation

   Distortion, deformation, stump formation and debris accumulation along the fibers are characteristics of the final stage of degeneration---degradation (Fig 1c,d). All were observed, mostly, at the late stage of 4 or more days after MA injection, following fiber varicosis, perforation, and fragmentation. The 5-HT fiber tracts contained larger number of deteriorated varicose fibers and terminal stumps. Degradation occurred where fiber fragmentation was seen and at dosages of 25-50mg/kg. Many degenerated 5-HTT-im fibers were scattered throughout the cingulum bundle, septal nucleus, fornix, frontal and parietal cortices and hippocampus regions. The varicoses along the axons subsided, or degraded along with axonal fibers. The remaining 5-HTT-im fibers in the cortex were those of thick diameter (Figure 3b). It seems the thick 5-HTT-im fibers were either more resistant to the MA, or were thickened as a result of swelling process at this short-term observation point.

   Figure 3: Methamphetamine injection caused degeneration of 5-HTT-im fibers (b,d) as compared with saline injection (a,c) in the terminal regions--parietal cortex (a,b) and striatum (c,d). The dark-field microscopic photographs show dense serotonin fibers in layers I-III as indicated by 5-HTT-im staining. They are densely distributed in layer I near the surface of the cortex, and slightly less dense in layers II and III. Four days after MA (50 mg/kg) injection, the 5-HTT-im fibers degenerated across all layers. Layers I-III are shown in (b). Among the thick and thin type 5-HT fibers in the normal cortex, the remaining and more resistant 5-HTT-im fibers after MA injection seem to be thick fibers scattered throughout the cortex. The 5-HTT-im fibers were homogeneously distributed throughout the striatum in the saline injected control rat brain. Four days after MA injection, very few 5-HTT-im fibers remained; dilated, degenerating 5-HTT-im fibers were still seen (d, arrows). Scale bars: a,b,c,d=80µm

    

Time course versus dosages
The two phases of 5-HT fiber damage are both time and dosage dependent. The first phase occurred within hours; varicosis occurred at dosages of 12.5-50mg/kg, perforation was seen from 25 to 50mg/kg, and disintegration at 50mg/kg only. The second phase occurred from 2.5 hours to days; where varicose-perforation occurred fragmentation and degeneration prevailed. Substantial fiber loss (Fig. 4) occurred as a result of either disintegration or degeneration in each phase, depending on the dosage.

Regional consideration
Wide regions of the brain were affected by the MA injections. It seems the dorsal side of the brain was more affected than the ventral side. The 12.5mg/kg dosage mainly affected the frontal cortex and hippocampus. Among the 25-50mg/kg MA injected animals, the most affected regions were the frontal and parietal cortices, striatum (Fig. 3), hippocampus (Fig. 4), habenula nuclei and dorsal thalamus where 5-HTT-im fibers were reduced compared to their usual density. The degree of fiber loss was dosage dependent. An example is shown in the cortex (Fig. 5). The major 5-HT fiber pathways, medial forebrain bundle, cingulum bundle, indusium grisium, fornix, and stria terminalis, were also the targets of MA insult. Varicosis prevailed. Of the residual 5-HTT-im fibers, many were fragmented, torturous, and covered with degradation debris. The temporal cortex, hypothalamus and brainstem are relatively more resistant to MA insult. The 5-HTT-im fibers which survived in the temporal cortex, septal nucleus, nucleus accumbens, hypothalamus were mostly thick fibers where thick (2-3µm) and thin fibers (0.5-1µm) normally exist. The damage to 5-HTT-im and 5-HT-im cell bodies in the raphe (not shown), if it occurred, was largely unnoticeable over the current time course and dosages.

DISCUSSION

Definitive degeneration
A number of approaches are available for examining neuronal degeneration independent of transmitter level. The following markers can reveal neuronal profiles and are used to examine degeneration: (a) cytoskeletal marker (eg., neurofilaments), (b) neuronal impregnation marker (eg., silver impregnation), and (c) membrane marker (eg., Thy-1). With these markers, many morphological profiles--twisting fibers, terminal stumps, cellular debris, and the disappearance of fibers profiles are seen. The Fink-Heimer method of silver impregnation has been used to evaluate N, N-dimethylamphetamine and MDMA (Ricaurte et al., 1989; Slikker et al., 1988; Commins et al., 1986). To examine the degeneration of 5-HT neurons with 5-HT depleting drugs, a probe must satisfy additional criteria. The probe should be: (a) specific to 5-HT neurons, (b) non-diffusible, (c) presynaptic, and (d) capable of revealing morphological details.

5-HTT is a plasma membrane protein which mediates the uptake of 5-HT into presynaptic terminals following 5-HT release and action on a receptor. Our antibody against 5-HTT met the above criteria. Using 5-HTT antibody staining to detect 5-HT fibers, we confirmed that at doses of 12.5-50 mg/kg, MA caused definitive morphological changes (varicosis, perforation, and twisting fibers) and damage (disintegration, fragmentation, terminal stumps formation..etc) of 5-HT fibers. A reduction in the number of 5-HTT-im fibers results.

The 5-HTT staining revealed previously unseen damage to 5-HT fibers
The varicosis and perforation were uniquely revealed by 5-HTT staining perhaps because the 5-HTT-im localizes a membrane structure which outlines the 5-HT fibers, while 5-HT-im staining reflects the content of the 5-HT fibers, dependent upon the existing or detectable amount of 5-HT within the fibers. When 5-HT is deprived as a result of either a pharmacological depletion or a physical leak through perforation as the result of drug insult, 5-HT-im may fail to reveal the fiber profile due to the lack of 5-HT in the fibers regions, while 5-HTT-im can show the deterioration of the 5-HT fiber profile.

Two phases of 5-HT fiber destruction
We report here, for the first time, that the 5-HT fiber deterioration process occurs in two phases: (1) Acute Phase --Varicosis, Perforation, and Disintegration---depending on the dose of MA (Fig. 6). Within hours of administration of 12.5-50mg/kg of MA , the 5-HT fibers formed varicoses of various sizes. The varicoses may have enlarged when continuously exposed to MA. Varicose enlargement exceeding the capacity of membrane stretch may have resulted in perforation and disintegration. In the high dose (50 mg/kg/day) MA group, the 5-HTT-im fiber density already exhibited minor reduction in the frontal, parietal and cingulate cortices, hippocampus, and thalamic regions at the time of examination. The 5-HT fibers and / or 5-HT transporter might have decomposed as a result of the severe MA insult. This also indicates that (a) there is deferential sensitivity, and (b) that a small population of 5-HTT-im fibers is very sensitive to MA injection and disintegrates within two and half hours. (2) Chronic Phase--Fragmentation and Degradation. Within 4 days after MA injection, 5-HT fibers became fragmented at the perforated site, and were undergoing anterograde and / or retrograde degradation. The distinctive feature of this phase is the breakdown of the fibers with various shape of debris through the late degenerative process or, conversely, the recovery of the perforated 5-HTT-im fibers. Another feature was the reduction of the fiber number over larger areas [than in Phase I] of the brain.

Acute disintegration versus Chronic degradation
Disintegration may differ from degradation in morphology, time course, and specific mechanism. Degradation features prominent degeneration profile such as distortion, deformation, stump formation and debris accumulation, while disintegration contains few of the above but disappearance of fibers. Degradation occurs in chronic phase of degeneration, while disintegration in acute phase. Methamphetamine's intensive disintegration effect at 50mg/kg may be an acceleration of the decomposition process. It is likely that severe varicosis and perforation occur in a very short period of time (less than 2.5 hours) and 5-HT fiber disintegration occurs within hours. Supporting this notion was our observation of small number of terminal stumps (which are the result of characteristic chronic fiber degradation) during the first phase, though on a very limited scale. This could be a result of insult from superoxide radicals (Hirata et al., 1995) generate by MA or its secondary reactants. On the other hand, in Phase II, the fragmented 5-HT fiber segments degraded over a period of days, perhaps due to the lack of transportation of nutrient and vital protein supplies. At this stage, abundant degeneration profiles prevailed throughout the regions. In this regard, the rapid disintegration is unlikely to have been a result of the lack of vital nutrients.

The mechanism of MA damage to 5-HT fibers is unknown. It is hypothesized that the release of DA is an intermediate step in the cause of 5-HT degeneration (Johnson et al., 1978; Sonsalla et al., 1986). Blocking dopamine synthesis prevents 5-HT degeneration (Stone et al., 1989; Schmidt et al., 1985). We observed a large reduction of 5-HTT-im fibers in the hippocampus in both the first and in the second phases. Very few DA fibers are known to be located in the hippocampus, and the high norepinephrinergic innervation does not mediate the 5-HT damage (Johnson et al., 1991). The involvement of excitatory transmitters and the alteration of body temperature on toxicity of 5-HT fibers needs to be studied in the future. The study of the relationship between dopamine release and damage to 5-HT fibers, using 5-HTT antibodies, is under investigation.

In conclusion, using 5-HTT antibody staining to detect 5-HT fibers, we confirmed that 25-50 mg/kg dose of MA caused definitive damage and degeneration of 5-HT fibers in the frontal, parietal and cingulate cortices, hippocampus, and thalamic regions. The thin 5-HTT-im fibers seem to be more vulnerable than the thick fibers. Damage to 5-HT cell bodies was unnoticeable. In addition, we report here, for the first time, that there are two sequential phases of 5-HT fiber destruction: Phase I, Varicosis at dosages of 12.5-50m/kg, perforation at 25-50m/kg, and disintegration at 50 mg/kg, which occurs within hours; Phase II, fragmentation and degeneration, which takes place within days. Damage to 5-HT fibers is dose dependent between 12.5-50 mg. Reduction of 5-HT fibers in the brain occurs and is likely to result in long-term consequences on hypo-serotonin functions or behavior.

ACKNOWLEDGMENTS

This study is supported by NIH R24HD30508 to F.C.Z. The authors thank Miss Janet Donaldson for editing the manuscript.

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