BDNF increases monoaminergic activity in rat brain following intracerebroventricular or intraparenchymal administration

BRAIN RESEARCH ELSEVIER

Brain Research 71(l (1996) 11-20

Research report

BDNF increases monoaminergic activity in rat brain following intracerebroventricular or intraparenchymal administration Judith A. Siuciak *, Carolyn Boylan, Michelle Fritsche, C. Anthony Altar, Ronald M. Lindsay Regeneron Pharmaceuticals, 777 Old Saw Mill Rit~er Road, Tarrytown, N Y 10591, [.'SA

Accepted 3 October 1995

Abstract

We have previously demonstrated alterations in serotonin metabolism within descending pathways following infusion of brain-derived neurotrophic factor (BDNF) into the midbrain, near the periaqueductal gray and dorsal and median raphe nuclei. The aim of the present study was to extend these studies to include a comprehensive regional examination of monoamine (serotonin, dopamine and norepinephrine) and metabolite levels in discrete areas of the intact, adult rat forebrain following direct intraparenchymal midbrain BDNF infusion. We have compared neurochemical changes following midbrain infusion of BDNF to those obtained following intracerebroventricular (i.c.v.) infusion. Significant increases in levels of 5-HIAA and/or the 5-HIAA/5-HT ratio were found in all areas examined including the hippocampus, cortex, striatum, n. accumbens, substantia nigra and hypothalamus following both midbrain and i.c.v. infusion. Changes in dopaminergic activity were also observed, but displayed more regional specificity, i.e. changes were found primarily within the striatum and cortex. The two infusion sites produced similar patterns of neurochemical effects although the magnitude of the changes did vary in some areas. These results suggest that BDNF increased synthesis and/or turnover of serotonin, and to a lesser extent dopamine, in the mature rat forebrain. Furthermore, these data point to possible functional roles for BDNF in neuropsychiatric and neurodegenerative conditions which involve a dysregulation of these monoamine systems. Keywords: Brain-derived neurotrophic factor; Norepinephrine; Dopamine; Serotonin; Raphe nucleus: Periaqueductal gray: Hippocampus: Cortex: Nucleus

accumbens: Hypothalamus; Substantia nigra

1. Introduction

Brain-derived neurotrophic factor (BDNF) is a member of the nerve-growth factor (NGF) family of neurotrophic factors (for review, see Ref. [28]). Using in situ hybridization, m R N A for both BDNF and TrkB receptor tyrosine kinase, the high affinity receptor for BDNF, were found to be widely expressed throughout the rat forebrain, including the cortex, hippocampus, hypothalamus and other areas of the CNS [3,18,22,24,35,52,54]. Many studies have examined the influence of neurotrophic factors upon the survival and differentiation of

Abbreviations: BI)NF, brain-derived neurotrophic factor; DA, dopamine; DHBA, dihydroxybenzylamine; DOPAC, dihydroxyphenylacetic acid; 5-HT, serotonin; 5-HIAA, 5-hydroxyindoleacetic acid; HVA, homovanillic acid; i.c.v., intracerebroventricular; NE, norepinephrine: NGF, nerve growth factor; NRM, nucleus raphe magnus; NT-3, neurotrophin-3; NT-4/5, neurotrophin-4/5; PAG/DR, periaqueductal gray/dorsal raphe: VEH, vehicle-infused * Corresponding author. Fax: (1) (914) 345-7739; E-mail: j udy.siuciak(i~, regpha.com tX)06-8993/96/$15.00 © 1996 Elsevier Science B.V. All rights reserved SSDI 001)6-~9t,~3t 95 )(I I 289-3

various neuronal populations (for review, see Ref. [29]). However, there is rapidly growing evidence that BDNF has neuronal effects that are unrelated to survival in the intact, adult rat brain, i.e. modulatory effects on monoamines, neuropeptides and behavior (for review, see Ref. [29]). For example, in vivo BDNF administration has been reported to modulate serotonin [42,47], dopamine [2,26,33] and several, neuropeptide systems [11,37,50], as well as producing hypophagia and weight Joss [25,42,44], altered patterns of locomotor behavior [33], analgesia [47,50] and an antidepressant-like effect [48,49]. To date, a comprehensive regional examination of the neuromodulatory effects of exogenous BDNF on central monoaminergic systems has not been undertaken. The present studies were designed to explore BDNF-induced changes in monoamine (5-HT, DA and NE) and metabolite (5-HIAA, DOPAC, HVA) levels within various forebrain areas. We have compared two routes of administration, infusion into the lateral ventricle or directly into the midbrain. The midbrain infusion site, near the periaqueductal gray and dorsal and median raphe nuclei, permits BDNF

12

J.A. Siuciak et al. / Brain Research 710 (1990) 11-20

access to the largest number of serotonergic cell bodies in the brain. The ascending projections of the dorsal and median raphe nuclei include the hippocampus, cerebral cortex, striatum and hypothalamus [51]. In contrast to the localized midbrain delivery, infusion of BDNF into the lateral ventricle permits BDNF access to a wide range of periventricular areas, including, direct, albeit limited, access to forebrain areas such as the striatum, hippocampus and hypothalamus [4,58]. These studies provide insight into the mechanisms by which BDNF modifies behavior in intact rats and provide information on possible novel roles for neurotrophic factors in the adult central nervous system.

tional landmarks [41]. The samples were stored frozen until assay. Tissues were homogenized in 10 vols. of 0.32 M sucrose. A 90-/xl aliquot for monoamine determinations was added to 10 /xl of a 4 N perchlorate, 1 mM ascorbate, 3 /zg/ml dihydroxybenzylamine (DHBA) internal standard solution. Aliquots of the sucrose homogenate were removed for protein determination (Pierce BCA protein assay). Serotonin (5-HT), 5-hydroxyindoleacetic acid (5HIAA), norepinephrine (NE), dopamine (DA), dihydroxyphenylacetic acid (DOPAC), and homovanillic acid (HVA) were measured using an isocratic HPLC elution system and electrochemical detection, using a 16-channel coulometric array detector (ESA, Inc., Bedford, MA) and established methods [16].

2. Materials and methods

2.3. Statistical analysis

2.1. Subjects" and surgery

Statistical significance of the body weight data from midbrain and i.c.v, infusions studies was assessed using a repeated measures ANOVA followed by a Scheffe's post hoc test. For the midbrain infusion studies, analysis of HPLC data from VEH- and BDNF-infused rats was performed using an unpaired Student's t-test with P < 0.05 taken as significant. For analysis of the HPLC data from i.c.v, infusions, a two-way ANOVA was performed comparing treatment (VEH, VEH-FD or BDNF) and side (infused vs uninfused), with the exception of the n. accumbens data, where only the right (infused) side was used due to degradation of the left side tissues. No statistical differences were found between the infused/uninfused sides, thus the HPLC data for the two sides were averaged and these data were then reported in the table and analyzed for statistical significance with a one-way ANOVA. When F ratios were significant ( P < 0.05), Scheffe's test of multiple comparisons was used for post-hoc analysis with P <; 0.05 considered significant. The commercial computer software, SuperANOVA (Abacus Concepts Inc., Berkeley, CA) was used for all statistical analyses.

Experiments were approved by the Animal Care and Use Committee at our institution and were conducted in compliance with NIH guidelines. Male Sprague-Dawley rats (300-350 g) were used and surgery was performed as previously described [47,50]. For midbrain infusions, 6.8mm long cannulae were implanted into the midbrain, near the periaqueductal gray and dorsal raphe nucleus ( - 7 . 6 mm from bregma, 1 mm lateral from the sagittal suture). For unilateral i.c.v, infusions, 4.2-mm long cannulae were implanted into the lateral ventricle ( - 0.4 mm from bregma, 1.8 mm lateral from the sagittal suture). Each cannula was attached via a 2-cm length of tubing to an osmotic pump (Alzet model 2002) which was implanted subcutaneously between the shoulder blades. Animals received either phosphate buffered saline (VEH, 12 /xl/day) or BDNF (12-115 /zg/day for midbrain infusions or 2 4 / x g / d a y for i.c.v, infusions). For midbrain infusion studies, only animals receiving 12 /xg/day BDNF were used for neurochemical analysis, higher doses were used to further examine the effects of midbrain BDNF on body weight. For the i.c.v, studies, an additional group of vehicle-infused rats were subjected to food restrictions (VEH-FD) such that body weights would be similar to those of the BDNF-infused rats during the experiment. Except for weight loss or lack of weight gain, animals appeared healthy and no significant morbidity or mortality was observed.

2.2. Neurochemical measurements At 12-14 days after surgery, the animals were sacrificed and the brains removed. The hypothalamus was removed using a curved forceps, then each brain was cut into sequential, 1.5-ram-thick coronal slabs. Each slab was placed on a metal block kept on ice. The hippocampus, frontal/parietal cortex, neostriatum, nucleus accumbens, substantia nigra, and locus coeruleus were dissected with either a micropunch or scalpel blade according to conven-

3. Results

3.1. Midbrain infusions 3.1.1. Body weight The effect of midbrain infusion of BDNF (12, 60, 115 /zg/day) on body weight is shown in Fig. 1. A repeated measures ANOVA indicated an overall effect of BDNF concentration (F3, % = 6.7, P = 0.0012), and duration of infusion (Fx9 ~ = 34.9, P = 0.0001). Furthermore, a significant interaction between infusion time and treatment was found (F,,~6 = 17.9, P < 0 . 0 0 0 1 ) such that increased BDNF concentration resulted in greater weight loss. Posthoc analysis showed no significant weight changes following the 12 /xg/day dose ( P = 0.83), but higher doses did produce significant effects on body weight (60 /zg/day,

J.A. Siuciak et al. ~Brain Research 710 11096) 11-20 MIDBRAIN --~]--•

INFUSION

13

lose weight by day 5 and body weights appeared to stabilize between days 5 and 11.

VEH BDNF 12 ug/day

- ~

B D N F 60 ug/day

~---

3.1.2. Neurochemical effects"

BDNF 115 ug/day

E

The levels of monoamines and their metabolites measured within the brains of intact, adult rats following midbrain infusion of BDNF (12 / z g / d a y ) are shown in Table 1.

I."1L9 m LU

3.1.3. Serotonergic system

I21 O m

2

i

7

5

9

10

11

12

TIME AFTER ONSET OF INFUSION (days)

Fig. 1. Effect of midbrain infusion of BDNF on body weight. Rats received infusions of BDNF (12, 60 or 115 p . g / d a y ) or PBS vehicle into the midbrain, near the periaqueductal gray and dorsal raphe nucleus for 14 days. Values shown are means i S.E.M. for 6 - 1 2 r a t s / g r o u p .

P < 0.04; 115 # g / d a y , P < 0.007). All infusion groups showed a slight weight loss on the day after surgery (day 1), although, after that time, the VEH-infused rats began to gain weight, whereas the BDNF-infused animals exhibited a delayed recovery at lower doses and weight loss at higher doses. By day 11 of infusion, BDNF-infused rats receiving the highest dose (115 p~g/day) weighed 33% less than the VEH-infused rats (VEH, 369 _+ 9 g vs BDNF, 245 + 14 g, P < 0.007). Even at the highest concentration of BDNF (115 /xg/day), midbrain-infused rats ceased to

An augmentation in serotonergic activity was found in all brain areas examined. Increased levels of serotonin were found only in the cortex (21% increase, t = 2.28, df = 13, P < 0.04). Levels of 5-HIAA, the major metabolite of 5-HT, were elevated in all brain areas, including the hippocampus (67%, t = 3.8, P < 0.002), striatum (57%, t = 4.2, P < 0.001), cortex (55%, t = 4.5, P < 0.001), and substantia nigra (53%, t = 3.5, P < 0.0114). The nucleus accumbens showed a trend towards increased 5-HIAA levels (60%, P < 0.06), but no change was seen in the hypothalamus (t = 0.32, P = 0.76). Increased serotonin turnover, as indicated by an increase in the 5 - H I A A / 5 - H T ratio, was found in all 6 brain areas examined, with the hippocampus (63%, t = 3.8, P < ().0021 and substantia nigra (66%, t = 3.6, P < 0.004), showing the greatest percentage change. Although no significant changes in 5-HT or 5-HIAA were seen in the n. accumbens and hypothalamus, there were modest increases in the 5H I A A / 5 - H T ratio in these regions in. accumbens, 337~, t = 2.5, P < 0.03; and hypothalamus, 27%, t = 2.9, P < 0.011.

Table 1 Levels of monoamincs and their metabolites measured within the brains of intact, adult rats following midbrain infusion of BDNF (12 p , g / d a y ) or PBS vehicle 5-HT (pg/#g protein) Hippocampus Vehicle 2.6 ~ BDNF 2.6 * Cortex Vehicle 1.9~ BDNF 2.34 Striatum Vehicle 2.4~ BDNF 2.(~ Nucleus accumbens Vehicle 6.t~ ~ BDNF 7,2 ~ Substantia nigra Vehicle 105~ BDNF 10 1 q Hypothalamus Vehicle 96~ BDNF 6.(~ ~

5-HIAA (pg//~g protein)

5-HIAA/5-HT ratio

Dopamine (pg//xg protein)

DOPAC Ipg//xg protein)

DOPAC/DA ratio

HVA (pg/,ag protein)

HVA/I)A ratio

NE (pg/#g protein)

0.2 11.2

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0.98 _+ 0.08 1.59 ± 0.15 "

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0.45 + 0.05 11.58 _+ 0.1)8

n.d. n.d.

n.d. n.d.

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0.60±0.02 0.79±0.04 "

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n,d. n,d.

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n.d. n.d.

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2.2 0.5

6.3-+ 1.3 5.8 ± 0.8

Values shown are means ± S.E.M. for 7 - 8 r a t s / g r o u p , n.d., not done. " Significantly different from vehicle, Students's t-test, P < I).05.

1.5 + I). 1 1.5 + I).l n.d. nd.

J.A. Siuciak et al. / Brain Research 710 (1996) 11-20

14

3.1.4. Dopaminergic system No changes in dopamine levels were found in any of the brain areas examined, whereas elevations in the DA metabolites, DOPAC and HVA, were found within the cortex a n d / o r striatum. In the cortex, the D O P A C / D A ratio was increased by 33% (t = 2.3, P < 0.04) and levels of HVA and the ratio of H V A / D A were increased by 54% (t = 4.7, P < 0.001) and 55% (t = 2.4, P < 0.03), respectively. The striatum showed the greatest changes in dopaminergic activity, with DOPAC levels increased by 33% (t = 3.7, P < 0.003) and the D O P A C / D A ratio by 25% (t = 2.9, P < 0.01). In addition, HVA levels and the H V A / D A ratio were elevated by 73% (t = 5.7, P < 0.001) and 63% (t = 4.0, P < 0.001), respectively. Due to technical problems, at the time of assay, HVA levels could not be determined in the samples of hippocampus, n. accumbens, substantia nigra or hypothalamus.

3.1.5. Noradrenergic system No changes in norepinephrine levels were found in any of the brain areas examined.

3.2. IntracerebroL,entricular infusion 3.2.1. Body weight Since considerable weight loss has been reported following i.c.v. BDNF administration [25,42,44], food-deprived VEH-infused rats were run concomitantly as additional controls. The effect of i.c.v, infusion of BDNF (24 /,tg/day) on body weight is shown in Fig. 2. A repeated measures ANOVA indicated a significant effect of treatment on body weight (F2,24 = 41.54, P < 0.0001) such that both BDNF infusions ( P < 0.001) and food deprivation in VEH-infused rats ( P < 0.001) resulted in a significant loss of body weight versus VEH-infused rats which were allowed food ad libitum. By day 14 of infusion,

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3.2.2. Neurochemical effects The levels of monoamines and their metabolites within the brains of intact, adult rats following unilateral ventricular infusion of BDNF is shown in Table 2.

3.2.3. Serotonergic system An increase in serotonergic activity following i.c.v. BDNF administration was observed in all brain areas examined. No significant changes in levels of 5-HT were observed in any of the brain areas examined. Increased levels of 5-HIAA were found in all brain areas including the cortex (139% vs VEH, P < 0.0001), n. accumbens (98% vs VEH, P < 0.0006), locus coeruleus (82% vs VEH, P < 0.03), striatum (77% vs VEH, P < 0.0001), substantia nigra (66% vs VEH, P < 0.0001), hippocampus (48% vs VEH, P < 0.003), and hypothalamus (36% vs VEH, P < 0.01). Increased serotonin turnover, as indicated by the increased 5 - H I A A / 5 - H T ratio, was found in all brain areas (except for the locus coeruleus), with the percentage increase ranging from 42% (hypothalamus) to 87% (cortex). No differences in serotonin or metabolite levels were observed between non-food-deprived and food-deprived, vehicle-infused rats with the exception of the hypothalamus, where serotonin levels in food-deprived rats were decreased in comparison to rats allowed ad libitum access to food (22% decrease, P < 0.02). This decrease is 5-HT was not accompanied by significant alterations in the levels of 5-H1AA or the 5-HIAA/5-HT ratio within the hypothalamus.

3.2.4. Dopaminergic system

INFUSION

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BDNF-infused rats weighed 33% less than the VEH-infused rats (VEH, 458 + 6 g; vs BDNF, 305 + 24 g, P < 0.0001). No differences in weight were found between BDNF-infused and food-deprived VEH-infused rats (VEHFD, 322 + 4 g vs BDNF, P = 0.12).

,6

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7

8

9

10

11

OF INFUSION

12

~

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(days)

Fig. 2. Effect of unilateral i.c.v, infusion of BDNF on body weight. Rats rcccived unilateral infusions of BDNF (24 /xg/day) or PBS vehicle into the right lateral ventricle for 14 days. Values shown are means + S.E.M. for 8-16 rats/group.

The effect of i.c.v. BDNF infusion on dopaminergic activity was region specific in that changes were found primarily in the cortex, where dopamine levels were increased by 30% (vs VEH, P < 0.04). Increases also were seen in both metabolites, DOPAC (100% vs VEH, P < 0.0001) and HVA (120% vs VEH, P < 0.007) and in the D O P A C / D A (45% vs VEH, P < 0.003) and H V A / D A (66% vs VEH, P < 0.03) ratios. Within the hippocampus, no effects on DA levels were observed; however, DOPAC (60% vs VEH, P < 0.0001) and the D O P A C / D A ratio (60% vs VEH, P < 0.0001) were significantly increased and there was a trend towards increased HVA levels ( P < 0.057). Conversely, the striaturn showed alterations only in HVA (68%, P < 0.0008) and the H V A / D A ratio (66%, P < 0.003) with no effects seen for DA or its other metabolite, DOPAC. In the locus coeruleus, BDNF infusion significantly increased levels of

J.A. Siuciak et a L / Brain Research 710 ( 19901 11-20

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J.A. Siuciak et al. /Brain Research 710 (1996) l 1-20

DA, DOPAC and HVA by 118% ( P < 0 . 0 2 3 ) , 214% ( P < 0.0021) and 154% ( P < 0.009), respectively, while the ratios of metabolite/DA did not change. Within the n. accumbens, H V A / D A ratios were increased by 53% vs VEH-infused rats ( P < 0.02), no additional changes in DA, DOPAC or HVA levels as a result of BDNF infusion were found in this area. Only the substantia nigra and the n. accumbens showed changes in dopaminergic activity between non-food-deprived and food-deprived VEH-infused rats. In the nigra, HVA levels were significantly decreased (FD vs VEH: 34% decrease, P < 0.04). The nigral HVA levels in BDNF-infused rats were not significantly different from those obtained in food-deprived vehicle-infused rats ( P = 0.96). This decrease in HVA was not accompanied by significant changes in DA and DOPAC levels or in the H V A / D A ratio. In the n. accumbens, levels of the metabolites and their ratios were significantly increased (FD vs VEH: DOPAC, 54%, P < 0.04; D O P A C / D A , 39%, P < 0.03; HVA, 75%, P < 0.03; H V A / D A , 55%, P < 0.02). Within the n. accumbens, the levels of these compounds following i.c.v, infusion of BDNF were not significantly different from those obtained in food-deprived vehicle-infused rats ( P > 0.2).

3.2.5. Noradrenergic system BDNF infusion increased levels of NE by 29% in the cortex ( P < 0.003), 89% in the locus coeruleus ( P < 0.03), and 102% in the n. accumbens ( P < 0.003). No other changes in NE were found in any of the additional brain areas examined. No differences in norepinephrine were found between non-food-deprived and food-deprived vehicle-infused rats in any brain area examined.

4. Discussion

The present report demonstrates that BDNF, infused directly into the intact, adult rat brain, produces regionspecific changes in the activity of monoaminergic systems in the forebrain areas examined. These studies extend our previous findings of an augmentation of serotonergic activity at the site of infusion (PAG/dorsal raphe) and within descending pathways (NRM and spinal cord) following infusion of BDNF into the midbrain. Furthermore, these studies demonstrate that midbrain and i.c.v. BDNF administration produced many similar neuromodulatory effects on central monoaminergic systems.

4.1. BDNF effects on body weight The route of BDNF administration proved to be an important factor with respect to BDNF effects on body weight. Rats infused chronically with BDNF into the lat-

eral ventricle showed a pronounced behavioral syndrome of reduced food and water intake and body weight loss as previously reported [25,42,44]. l.c.v, infusion of BDNF had a greater effect on body weight than the midbrain route of administration. It required an almost 5-fold greater amount of BDNF infused directly into the midbrain to produce an effect on body weight equivalent BDNF delivered i.c.v. (24/xg/day). However, even at the highest dose (115 /xg/day) animals ceased to lose weight by day 5 following initiation of midbrain infusion, whereas rats receiving i.c.v, infusions (24 /xg/day) continued to show weight reductions until the time of sacrifice. Neurochemical analysis was performed in midbrain-infused rats receiving 12 /,xg/day of BDNF, which produced a minimal effect on weight and in rats receiving 24 /xg/day BDNF i.c.v., which had a profound effect on body weight. Pelleymounter et al. [42] suggested that ventromedial hypothalamic transmitter systems, in particular 5-HT, may mediate the effects of BDNF on appetite and body weight. Electrical stimulation of the ventromedial hypothalamus or infusion of 5-HT in this area has been reported to suppress eating [27]. The present studies demonstrate an increase in serotonergic activity as indicated by increased 5-HIAA content and 5 - H I A A / 5 - H T ratio within the hypothalamus following i.c.v, infusion of BDNF. A lesser effect was seen on hypothalamic 5-HT activity following midbrain infusions of BDNF, which correlates well with the decreased effect of midbrain BDNF on body weight. Furthermore, the only alteration in 5-HT activity seen in VEH-infused food-deprived rats was a decrease in hypothalamic 5-HT levels as compared to VEH-infused rats allowed ad libitum access to food, indicating that the serotonergic effects of BDNF were not secondary to food deprivation. Thus, as suggested by Pelleymounter and colleagues, alterations in hypothalamic serotonergic activity may play a role in BDNF-induced hypophagia. Sauer et al. [44] suggested that dopamine was the causative agent of BDNF's effects on body weight, since treatment with dopamine-releasing drugs, such as amphetamine, produce aphagia very similar to that observed in BDNF-infused animals. In the present studies, the i.c.v. route of administration produced both a greater effect on weight loss and dopaminergic activity, particularly in the cortex and n. accumbens, as compared to the midbrain infusion. Future studies to determine the site and mechanism of the hypophagic action of BDNF should examine differential effects of BDNF on body weight in relation to the site of infusion (i.e.i.c.v., hypothalamus, etc.) in intact animals and animals which have sustained selective lesions of the implicated neurotransmitter systems using neurotoxins such as 6-hydroxydopamine (6-OHDA) and/or 5,7-dihydroxytryptamine (5,7-DHT). Furthermore, although these results suggests the possible contribution of both dopaminergic and serotonergic systems, these studies do not rule out mechanisms independent of these systems.

J.A. Siuciak et al. / Brain Research 710 (1996) l 1-20

4.2. BDNF effects on serotonergic systems Infusion of BDNF by both routes of administration produced similar neurochemical changes in that a dramatic augmentation in the activity of the 5-HT system was seen in all brain areas examined following either route of BDNF administration. Increases in the levels of 5-HT itself were limited to the cortex and these were obtained only in the midbrain infusion experiments. However, elevated 5-HT turnover was observed in all brain areas. There were some regional differences in the magnitude of changes observed using the two infusion sites. For example, the greatest increase in serotonergic activity seen following midbrain brain infusion of BDNF was in the hippocampus (5-HIAA = 67%, 5 - H I A A / 5 - H T = 63% increase), while the i.c.v, route produced the greatest effects in the cortex (5-HIAA = 139%, 5 - H I A A / 5 - H T = 87% increase). After midbrain infusions, hypothalamic 5-HIAA was not changed and the 5 - H I A A / 5 - H T ratio was only minimally increased (27%), however, i.c.v, administration resulted in a greater effect on hypothalamic serotonin metabolism and turnover (5-HIAA = 36% and 5 - H I A A / 5 - H T ratio = 63% increase), possibly due to access of BDNF administered i.c.v, to this periventricular structure [4]. The present results showing increases in hypothalamic 5-HIAA and 5H I A A / 5 - H T , without concomitant changes in hypothalamic 5-HT levels following i.c.v.-infused BDNF agree with those recently reported by Pelleymounter and colleagues [42]. Several studies have reported changes in serotonergic activity following central BDNF administration. BDNF infused into the midbrain, near the periaqueductal gray and dorsal raphe nucleus, increased levels of serotonin a n d / o r its primary metabolite, 5-HIAA, at the site of infusion as well as in descending pathways within the n. raphe magnus and lumbar spinal cord [47]. Rats receiving midbrain infusions of BDNF were analgesic in the tail-flick, hot-plate and formalin tests [47,50]. Increased 5-HT turnover in the striatum of rals receiving unilateral infusion of BDNF into the substantia nigra has also been reported [33]. In addition, we have previously reported the use of cytochrome c as a protein control to demonstrate the specificity of the effect of BDNF on serotonergic systems [30]. The mechanism of BDNF's modulatory effects on serotonin activity is unclear at the present time. BDNF has recently been reported to have no effect on [3H]5-HT uptake in vitro or ex vivo [30]. White and colleagues [56], using immortalized serotonergic neurons, have reported that BDNF enhanced levels of tryptophan hydroxylase, the rate-limiting enzyme in the synthesis of serotonin.

4.3. BDNF effects on dopaminergic systems The effects of BDNF on dopaminergic activity were more regionally restricted than that seen for the serotonin system. The two routes of administration produced similar

17

effects neurochemically, in that increases in dopaminergic activity were primarily limited to the cortex and striatum. However, as observed with the serotonin system, the magnitude of these changes varied between the two infusion sites. In the cortex, i.c.v, infusion increased DA levels by 30% and produced a much larger effect on metabolite levels than the midbrain route of administration. In the striatum, both routes resulted in similar increases in HVA levels and the H V A / D A ratio, however, changes in DOPAC and the D O P A C / D A ratio were observed only after midbrain infusion. Finally, dopamine metabolism in the hippocampus and n. accumbens were significantly increased following i.c.v., but not midbrain, infusion of BDNF. Several lines of experimental evidence support a role for BDNF in modulating dopaminergic neurons directly. The distribution of neurons expressing BDNF mRNA in rat ventral mesencephalon corresponds well to the distribution of dopaminergic cells in the substantia nigra, ventral tegmental area and retrorubral region corresponding to the A9, A10 and A8 monoamine cell groups, respectively, and many of these neurons are tyrosine hydroxylase positive [15,45]. BDNF has been shown to promote the survival and differential of nigral dopamine neurons in culture [19,20,23]. Dopaminergic neurons of the substantia nigra bind, internalize and retrogradely transport exogenous BDNF after their infusion in to the striatum [4,36,55]. Chronic infusions of BDNF above the substantia nigra elevate dopamine metabolism, as determined by increases in the DOPAC/dopamine and HVA/dopamine ratios as well as contralateral rotations in unlesioned rats challenged with amphetamine [2,33]. In contrast, Lapchak et al. [26] reported a down regulation of dopaminergic markers ([~H]dopamine uptake, [3H]mazindol binding, tyrosine hvdroxylase activity and dopamine content) in the striatum following chronic intranigral BDNF administration. The findings of elevated serotonin turnover throughout the forebrain in response to midbrain or i.c.v, infusions of BDNF suggest that some of the increases in dopamine metabolism and release reported here and observed previously [2,33] may also be mediated by serotonin. Indeed, there is a relatively dense serotonin innervation of the dopaminergic dendrites in the substantia nigra [10]. Elevations of striatal serotonin potentiate striatal dopamine release and induce locomotor and contraversive rotational responses [13,40,53]. Consistent with a serotonergic modulation of dopamine neurochemistry is the ability of selective 5-HT 2 receptor antagonists to block amphetamine-induced increases in striatai dopamine release and locomotor behavior [39] and the ability of serotonin agonists to elevate striatal dopamine release and metabolism [7,43]. It should be emphasized that the literature concerning the regulation of dopamine neurons by serotonin is quite complex, with findings also supporting inhibitory roles for serotonin. It is interesting to speculate that BDNF may shift the balance of serotonin-dopamine interactions in

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J.A. Siuciak et al. / Brain Research 710 (1996) 11-20

favor of serotonergic excitation of dopamine metabolism, release, and behavioral expression.

4.4. BDNF effects on noradrenergic systems Changes in norepinephrine levels were seen only after i.c.v, administration of BDNF (cortex, n. accumbens and locus coeruleus). Midbrain infusion, albeit at a lower concentration of BDNF, did not produce this same effect, a disparity likely to be due to differences in the distribution of BDNF using the two routes of administration. BDNF would have more direct access to the locus coeruleus, which lies in the floors and walls of the fourth ventricle, only following i.c.v, infusion. However, in the case of both serotonin and dopamine, BDNF produced its major effects on metabolism a n d / o r turnover rather than the level of the transmitter itself. Therefore, it is not possible to conclude from the present study whether BDNF might have a similarly broad effect on norepinephrine turnover, since we were unable to measure levels of the noradrenergic metabolites, MHPG or normetanephrine. Several studies have suggested interactions between BDNF and noradrenergic systems, i.e. BDNF mRNA is present in low levels in the locus coeruleus and is greatly increased levels following reserpine administration [46]. In tissue culture, NT-3 and N T - 4 / 5 promote the in vitro survival of presumptive noradrenergic neurons derived from fetal rat locus coeruleus [14]. Furthermore, the retrograde transport of BDNF to noradrenergic cell bodies has also been demonstrated by the accumulation of immunoreactive material for BDNF in neurons of the locus coeruleus following infusion into hippocampus and substantia nigra (personal communication, S.J. Wiegand).

4.5. Implications for neurotrophin functions in the CNS These results suggest that both i.c.v, and midbrain infusions of BDNF increase the synthesis and/or turnover of serotonin and dopamine in the rat forebrain. Furthermore, these data may help point to possible novel functional roles for BDNF in neuropsychiatric and neurodegenerative conditions which involve a dysregulation of these monoamine systems. Marked serotonergic pathology has been observed during aging and in Alzheimer's disease [1,5,6,12,21,38,57]. There are reports of decreases in concentrations of 5-HT a n d / o r 5-HIAA in several brain regions (post-mortem) or the CSF from Alheimer's patients (for review see Ref. [17]). Cell loss and the presence of tangles have also been reported in the raphe nuclei [1,21,31,32,57]. The present data demonstrates that BDNF augments the function of serotonin neurons. In addition, a recent study also suggests that exogenous BDNF can augment structural aspects of the serotonergic system as well. Mamounas and colleagues [30] have recently reported that BDNF administration induced a sprouting of mature, intact serotonergic fibers and

prevented the loss of cortical 5-HT axons following administration of the serotonergic neurotoxin, p-chloroamphetamine (PCA). Several lines of evidence suggest that dysfunction of the serotonin and/or dopamine systems are involved in depression (for reviews see Refs. [8,9,34]). We have recently reported an anti-depressant-like effect of midbrain-infused BDNF in both the forced swim test and learned helplessness following inescapable shock paradigms [48,49]. It may be that the increased activity of the serotonin and dopamine systems in the forebrain may contribute to the antidepressant-like effect of BDNF. In conclusion, the present study demonstrates that BDNF modulates the activity of monoaminergic systems in the intact, adult rat brain. The exact mechanism(s) for the known pharmacological actions of BDNF (e.g. hypophagia, weight loss, changes in motor behavior, analgesia and anti-depressant-like activity) is unclear, however, changes in monoamine metabolism may be at least partly involved in these effects.

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