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Effects of neurotensin on regional brain concentrations of dopamine, serotonin and their main metabolites
Neuropeptides (1990) l&169-178 @ Longman Group UK Ltd 1990
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Effects of Neurotensin on Regional Brain Concentrations of Dopamine, Serotonin and their Main Metabolites A. D. DRUMHELLER”,
M. A. GAGNi*,
S. ST-PIERREt
and F. 6. JOLICOEUR*
*Department of Psychiatry, Faculty of Medicine, University of Sherbrooke, Sherbrooke, QuBbec, Canada, JlH 5N4 and tlNRS, 345 Hymus Blvd., Pointe Claire, QuBbec, Canada H9R lG6. (Reprint requests to FBJI
Abstract-The effects of neurotensin, 7.5 or 3Opg, on concentrations of DA, DOPAC, (HVA), serotonin 5-HT and 5-HIAA were measured in 8 regions of the rat brain either 5 or 30 min following intracerebroventricular administration. Regions examined included the frontal cortex, striatum, nucleus accumbens, amygdala, septum, hypothalamus, ventral tegmentum and substantia nigra. Results indicate that both doses of neurotensin significantly elevated concentrations of dopamine in the striatum and amygdala 5 min following injection. The effects of the peptide on DOPAC and HVA were more pervasive and enduring, with significant increases in metabolite levels occurring in both mesolimbic and nigrostriatal terminal regions. In order to assess effects on turnover of dopamine, the ratios of each metabolic to dopamine concentrations were examined. Results indicate that, while the DOPAC/DA ratio was elevated in many regions, the HVA/DA ratio was increased in all regions examined. The effects of neurotensin on serotoninergic parameters were less pervasive and more variable, with both increases and decreases in 5-HT and 5-HIAA concentrations being observed. The effects of the peptide on 5-HIAA/5-HT were limited to the nucleus accumbens, where this ratio was increased, and the ventral tegmentum, where 5-HIAA/5-HT was decreased. These findings reveal that the effects of the neurotensin on dopaminergic transmission are more widespread than previously reported in that all major dopamine pathways are affected by the peptide. Also, the observed changes in the ratios of both DOPAC and HVA to DA suggest that neurotensin enhances the turnover. of this transmitter.
Introduction Several lines of evidence suggest that the tridecapeptide, neurotensin (NT) interacts directly or indirectly with central dopaminergic systems (1, Date received 5 September 1989 Date accepted 5 September 1989
2). Neurotensin-like immunoreactivity is present in many areas containing dopamine (DA) cell bodies or terminals (3, 4). The peptide has been shown to be intraneuronally co-localized with DA in several discrete regions of the brain (5), and direct synaptic contact between NT and DA neurons has also been suggested (6,7). Behaviour-
169
170 ally, NT has been shown to decrease the hyperactivity induced by pre- and postsynaptic pharmacological stimulation of dopaminergic transmission (8, 9). Neurochemical studies have also supported a DA/NT interaction. Following DOPA decarboxylase inhibition, NT injected either intracisternally or intracerebroventricularly, increases DA synthesis as measured by DOPA accumulation in both mesolimbic and meostriatal terminals (10, 11). Some data suggest that the observed increase in DOPA is a result of a direct action of NT on presynaptic inhibitory DA receptors (11). The effects of centrally administered NT on regional levels of DA and its major metabolites, dihydrophenylacetic acid (DOPAC) and homovanillic acid (HVA) have also been studied and the results indicate that the neurochemical effects of the peptide are varied, depending on the dose administered and the time course examined. When administered intracisternally (IC), DA levels remain unchanged in the striatum, olfactory tubercles, nucleus accumbens and hypothalamus 60 min after the administration of 3, 30 or 100 pg NT (lo), but a later report from the same group indicated that an intermediate dose of 6Ol.~gNT increased levels of DA in the olfactory tubercle when measured 60 min after IC injection (12). On the other hand, the same authors reported a transient increase in striatal DA concentrations 30 min after administration of 30 u,g NT (10). The effects of the same doses of NT on regional DOPAC levels were also examined in the same regions. Significant elevations in this metabolite were found in the hypothalamus, striatum and olfactory tubercles, generally with the highest dose (lo), and in the nucleus accumbens with a lower (60 p_g) dose (12). The effects of the peptide on regional HVA levels appear to be more consistent. Dose dependent increases in this metabolite were found in all tissues examined (10, 12). Finally, when NT was injected intraventricularly (IVT), similar elevations in DOPAC and HVA concentrations were found with doses ranging from 3 to 1OOk.g in both striatum and nucleus accumbens (11). The purpose of the present study was to further investigate the effects of NT on dopamine metab-
NEURQPEPTIDES
olism in several projection areas of major dopaminergic pathways including the frontal cortex, amygdala, septum, nucleus accumbens, corpus striatum and hypothalamus. Furthermore, the effects of the peptide on dopamine metabolism within the regions containing the cells of origin of the dopamine projection systems, the substantia nigra and the ventral tegmentum, were also examined. The changes in dopamine metabolism were assessed following the IVT administration of both 7.5 and 3Okg of NT injected either 5 or 30 min prior to sacrifice. Finally, using the same experimental paradigm, the effects of the peptide on serotonin (5-HT) and its main metabolite, 5hydroxyindoleacetic acid (5-HIAA) were also assessed. Results are expressed as regional concentrations of DA, DOPAC, HVA, 5-HT and 5-HIAA in ng/mg wet weight, as well as the ratios DOPAC/ DA, HVAIDA and 5-HIAAIS-HT found in each region. It has been suggested that the use of metabolite/transmitter ratios provides a simple, reliable and non-invasive measure of neurotransmitter utilization (13, 14, 15).
Methods Animals
Male hooded rats obtained from Canadian Breeding Farm (St. Constant, Quebec) and weighing between 250 and 300g were used. Animals were housed in a temperature controlled room having a 12 hr light/dark cycle. Under pentobarbital anaesthesia, indwelling stainless steel cannulae were implanted into the left lateral cerebra-ventricle. Procedure
Following 48 hr of recovery, animals were divided into six groups (n = 8). Three groups of rats received either saline, 7.5 pg or 30 kg neurotensin in a volume of 20 kg1 0.9% NaCl, injected over a 30 set period by means of a 5Opl Hamilton syringe, and were sacrificed 5 min after injection. The other 3 groups received the same treatments, but were sacrificed 30 min after injection. Following a sharp blow to the head, brains were rapidly removed and placed on the dissection block on dry ice and regions excised according the method of Heffner
EFFECTS OF NEUROTENSIN ON REGIONAL BRAIN CONCENTRATIONS
et al. (16). The following regions were dissected: frontal cortex, hypothalamus, amygdala, nucleus accumbens, corpus striatum, substantia nigra, ventral tegmentum and septum. Regions were frozen at -80°C until analysis. All regions were homogenized in 0.1 M HC104 containing 400 p,l 1.OM sodium bisulphite. Homogenization volume for the cortex and amygdala was 1.Oml, and for the other regions, 400(~1. Brains were centrifuged at 15000rpm for 1.5min, the supernatants removed, filtered and 80 ~1 of each sample injected onto the column. Biochemical analysis
The separation and quantification of amines, indoleamines and their metabolites were carried out using a previously described method (17). Briefly, the chromatographic system (all components by Beckman Instruments) consisted of 2 pumps, a controller and an injector fitted with a 1OOpJ sample loop. An Ultrasphere ODS 5 l.~ reverse phase column (4mm x 15cm) was protected by a short precolumn. The column eluate was monitored by an LC 2A electrochemical detector (Bioanalytical Sytems) equipped with a glassy carbon electrode and operated at an applied potential of 0.65 V with reference to a silver/silver chloride electrode. The primary eluting solvent was a mixture of 0.1 M sodium acetate and 0.02M citric acid, pH 4.2 containing 0.3mM ethylenediamine-tetraacetic acid (EDTA) and 0.05mM sodium octysulfate. The secondary solvent was methanol. Amines were separated using a linear gradient of 08% methanol over 8 min, starting 4 min after injection. Twenty two minutes into the separation the % methanol was decreased to 0 over 2 min and the column re-equilibrated with starting buffer for 10 min prior to re-injection. All operations were performed at ambient temperature at a flow rate of 1.5mYmin. Injection volumes for standards and brain extracts remained constant at 8Ou.l. All solutions were filtered through 0.2l.~ nylon membranes (Ultipor, Chromatographic Specialties Co.) and thoroughly degassed prior to use. Quantitation of sample amine concentrations was performed by direct comparison of sample peak heights to the peak heights obtained follow-
171
ing the injection of known quantities of DA, DOPAC, HVA, 5-HIAA and 5-HT and converting to ng/mg wet weight. Chemicals
Neurotensin was synthesized by the solid phase synthesis technique according to a previously described procedure in our laboratory (18). DA, DOPAC, HVA, 5-HT and 5-HIAA were purchased from Sigma. Sodium acetate, citric acid, sodium bisulphite, HC104, EDTA and methanol were purchased from Fisher. Sodium octyl sulfate was purchased as a 40% solution from ICN. Distilled water was redistilled in glass and passed through a Norganic cartridge (Waters Associates) prior to use. Statistical analysis
For each time, region and substance measured, data were analyzed by means of individual oneway ANOVA’s. Levels of the main factor included treatments (0.9% NaCl, 7.5 and 3O.Op,g NT). Differences between saline controls and NT treated animals were assessed by means of Dunnett’s tests. In all cases a difference was considered significant if it had a probability of random occurrence of less than 5%. Results Effects of NT on regional concentrations of dopamine, DOPAC and HVA Dopamine. The effects of neurotensin on regional concentrations of dopamine are shown in Table 1. The levels of dopamine were significantly elevated in the corpus striatum and the amygdala by both 7.5 (p < 0.01) and 3OFg (p < 0.05) NT 5 min after injection. No significant effects of neurotensin were seen 30 min following administration in any region. DOPAC.
The effects of neurotensin on regional concentrations of DOPAC are shown in Table 1. Significant increases in DOPAC content were observed in the corpus striatum, nucleus accumbens, amygdala and hypothalamus 5 min following ICV injection of either 7.5 (p’s < 0.05) or 30.0 (p’s < 0.01) Fg of the peptide. In the septum, DOPAC
:D
:D
0.08 0.02 11.74” 2.56 7.50 1.49 0.66” 0.15 0.79 0.22 0.47 0.15 1.02 0.15 0.90 0.45
5 min 7.5 0.05 0.01 0.57b 2.07 7.29 1.72 0.61b 0.08 0.67 0.25 0.41 0.08 0.81 0.28 0.89 0.36
30.0 0.11 0.04 8.12 1.72 6.43 0.94 0.45 0.06 0.58 0.13 0.34 0.06 0.79 0.13 0.95 0.22
Sal 0.11 0.06 9.15 0.94 6.83 1.45 0.54 0.07 0.53 0.20 0.37 0.07 0.75 0.20 1.37 0.50
30 min 7.5 0.08 0.04 9.27 1.78 7.20 3.45 0.55 0.12 0.60 0.17 0.36 0.12 0.97 0.54 1.14 0.39
30.0 0.07 0.03 1.66 0.21 2.14 0.47 0.11 0.03 0.28 0.07 0.13 0.03 0.39 0.09 0.42 0.11
Sal 0.06 0.02 2.37 0.29 3.10b 0.49 0.16b 0.03 0.27 0.09 0.22b 0.07 0.41 0.09 0.38 0.18
5 min 7.5 0.09 0.02 2.83” 0.84 3.50” 0.75 0.29” 0.11 0.47” 0.17 0.44” 0.05 0.47 0.15 0.54 0.18
30.0 0.09 0.06 1.62 0.44 2.52 1.12 0.10 0.03 0.28 0.07 0.17 0.08 0.43 0.23 0.44 0.15
Sal
DOPAC
0.05 0.01 1.54 0.45 2.19 0.34 0.14 0.03 0.21 0.09 0.13 0.03 0.37 0.19 0.38 0.10
30 min 7.5 0.09 0.03 2.24b 0.78 4.07b 1.19 0.16 0.06 0.34 0.10 0.28” 0.06 0.52 0.27 0.40 0.12
30.0
of Dopamine, DOPAC and HVA
Results expressed in ng/mg wet weight Significant differences from Saline treated animals as revealed by two tailed Dunnett tests: a p < 0.01; b p < 0.05.
Substantia Nigra Ventral Tegmentum
!D u SD
:D
:D
!D
0.07 0.03 8.25 1.69 6.18 1.29 0.49 0.06 0.62 0.17 0.38 0.06 0.91 0.17 1.97 0.21
Sal
DOPAMZNE
of neurotensin on regional brain concentrations
ED
Effects
Hypothalamus
Septum
Striatum Nucleus Accumbens Amygdala
COlpS
Frontal Cortex
Table 1
0.10 0.02 0.82 0.06 0.91 0.11 0.09 0.03 0.21 0.02 0.05 0.02 0.34 0.09 0.37 0.05
Sal 0.11 0.03 1.23” 0.14 1.15b 0.33 0.12b 0.02 0.19 0.06 0.07 0.01 OMb 0.33 0.33 0.25
5 min 7.5
0.12 0.04 1.21” 0.41 1.37” 0.22 0.18” 0.05 0.51” 0.27 0.15” 0.07 0.83” 0.38 0.43 0.13
30.0
30 min 7.5 0.09 0.03 0.92 0.26 0.83 0.12 0.10 0.05 0.12 0.03 0.06 0.02 0.16 0.07 0.34 0.26
Sal 0.08 0.02 0.77 0.21 0.87 0.22 0.07 0.04 0.18 0.05 0.07 0.02 0.28 0.09 0.40 0.24
HVA
0.07 0.02 lLKrb 0.34 1.26” 0.23 0.11 0.03 0.26b 0.05 0.08 0.03 0.45 0.08 0.45 0.08
30.0
EFFECTS OF NEUROTENSIN ON REGIONAL BRAIN CONCENTRATIONS
levels were significantly increased at 5 min by the higher dose of NT (p < 0.01). Thirty min following administration of 3Ol.~gNT, DOPAC levels were significantly increased in the corpus striatum, nucleus accumbens (p’s < 0.05) and hypothalamus (p < 0.01). HVA.
The effects of neurotensin on regional concentrations of HVA are shown in Table 1. Significant increases in HVA levels were found in the corpus striatum 5 min after the administration of each dose of the peptide (p’s < 0.01). The administration of either 7.5 (p’s < 0.05) or 30.0 (p’s < 0.01) pg NT 5 min prior to decapitation also resulted in elevated HVA concentrations in the nucleus accumbens, amygdala and substantia nigra. In the septum and hypothalamus, only the higher dose of the peptide increased HVA levels at this time period (p < 0.01). HVA levels were still elevated 30 min after the injection of 30.0 but not 7.5 u,g of NT in the nucleus accumbens (p < O.Ol), corpus striatum, septum and substantia nigra (p’s < 0.05).
nucleus accumbens (p < 0.05) and in the hypothalamus following the administration of 7.5 pg of the peptide. These results are shown in Table 2. SHZAA.
Tissue levels of 5-HIAA were significantly increased 5 min after the injection of 7.5 Fg NT in the nucleus accumbens (p c 0.05). When measured 30 min following injection, SHIAA levels were increased in the corpus striatum with the higher dose of the peptide (p < 0.05) and was decreased in the hypothalamus with the lower dose tested (p < 0.01) (Table 2).
Effects of NT on regional concentrations of 5-HT and 5-HIAA 5-HT. The concentration
of 5-HT was decreased 30 min following the injection of 3Ocl.gNT in the Table 2
Effects of neurotensin
173
Effects of NT on metabolitelneurotransmitter
as well as the HVA/DA ratios were significantly increased in the frontal cortex following 3O~g NT both 5 and 30 min after injection (p’s c 0.01) (Table 3). No changes were observed in the ratio of 5-HIAA/5HT. Corpus striatum : The DOPAC/DA ratio was significantly increased by the 3Okg dose 30 min following administration (p < 0.05). HVA/DA was increased 5 min after the injection of 30 kg NT (p < 0.05) (Table 3). No changes were observed in the ratio 5-HIAA-5-HT. Nucleus accumbens : The DOPACYDA ratio was significantly increased in this region following the administration of 3Opg
on regional brain concentrations
of serotonin and 5-HIAA
Serotonin Sal
Frontal Cortex Corpus Striatum
ED
0.40 0.09 0.55
5 min 7.5
30.0
Sal
.5-HIAA 30 min 7.5
0.47
0.38
0.26
0.10 0.51 0.06 0.66
0.08
0.11
0.42
0.40
0.33 0.04 0.46
0.09 0.70
0.15 0.52
0.06 0.67
Nucleus
+
0.14 0.64
Accumbens
SD
0.17
0.15
0.22
0.13
0.23
0.50 0.12 0.30 0.05 0.69 0.15 0.87 0.30 0.73 0.26
0.56 0.15 0.35 0.09 0.74 0.15 0.91 0.31 0.70 0.27
0.46 0.08 0.58 0.21 0.65 0.16 1.22 0.36 0.99 0.30
0.48 0.09 0.34 0.15 0.61 0.21 1.10 0.36 0.93 0.14
0.42 0.17 0.25 0.10 0.38b 0.18 0.79 0.16 0.90 0.48
ED
Amygdala :D Septum Hypothalamus Substantia Nigra Ventral Tegmentum
:D u SD ;D s”D
ratios
Frontal cortex. The DOPAUDA
30.0
Sal
5 min 7.5
0.29 0.05
0.23 0.11
0.41 0.08 0.31” 0.14 0.44 0.07 0.41 0.12 0.55 0.15 0.95 0.36 0.88 0.14
30.0
Sal
30 min 7.5
30.0
0.32
0.19
0.07
0.09
0.21 0.10
0.28 0.02
0.28 0.05
0.56
0.59
0.42
0.45
0.57
0.61b
0.17 0.62 0.20 0.41 0.12 0.42 0.09 0.68 0.22 0.77 0.33 1.15 0.34
0.18 0.86b 0.12 0.44 0.11 0.34 0.06 0.74 0.12 0.84 0.20 1.01 0.24
0.10 0.61 0.20 0.33 0.06 0.54 0.31 0.47 0.11 0.83 0.17 1.03 0.21
0.15
0.09
0.08
0.59
0.70
0.65
0.17 0.47 0.06 0.32 0.08 0.73 0.19 0.76 0.17 1.23 0.33
0.17 0.37 0.10 0.37 0.06 0.40a 0.08 0.63 0.09 1.35 0.29
0.21 0.49 0.12 0.38 0.09 0.73 0.22 0.64 0.20 0.96 0.16
Results expressed in ng/mg wet weight Significant differences from Saline treated animals as revealed by two tailed Dunnett tests: a p < 0.01; b p < 0.05.
ED
s”D
0.73 0.19 0.21 0.04 0.43 0.11 0.24 0.07 0.36 0.10 0.55 0.29 0.40 0.12 0.40 0.07
1.7LGb 0.66 0.26 0.08 0.53b 0.18 0.49b 0.17 0.66 0.17 0.56 0.23 0.50 0.08 0.63” 0.12
30.0 0.82 0.21 0.19 0.02 0.42 0.13 0.26 0.13 0.47 0.12 0.48 0.19 0.52 0.21 0.41 0.05
Sal
HVAIDA
0.61 0.17 0.17 0.04 0.33 0.07 0.27 0.08 0.39 0.17 0.37 0.14 0.41 0.13 0.31 0.12
30 min 7.5
on ratios of metabolite
1.62” 0.47 0.24b 0.05 0.61” 0.18 0.28 0.06 0.57 0.10 0.92b 0.41 0.73 0.56 0.34 0.06
30.0 1.32 0.35 0.10 0.02 0.14 0.02 0.15 0.03 0.33 0.04 0.15 0.05 0.36 0.06 0.36 0.09
Sal
to transmitter
1.26 0.30 0.11 0.02 0.15 0.03 0.19 0.05 0.28 0.12 0.16 0.07 0.39 0.12 0.74 0.39
5 min 7.5 2.69” 0.75 0.14b 0.03 0.19” 0.03 0.29” 0.06 0.82b 0.40 0.31b 0.11 0.54b 0.25 1.29b 0.85
30.0 0.80 0.25 0.10 0.02 0.15 0.04 0.15 0.06 0.40 0.40 0.21 0.06 0.34 0.05 0.35 0.09
Sal
5-HIAA
concentrations
0.92 0.28 0.11 0.03 0.13 0.07 0.18 0.05 0.27 0.05 0.15 0.04 0.26 0.08 0.25 0.03
30 min 7.5 1.83” 0.50 0.12 0.03 0.20 0.07 0.18 0.06 0.40 0.07 0.25 0.14 0.42 0.18 0.46 0.18
30.0 0.75 0.21 1.04 0.29 0.97 0.28 0.80 0.20 1.29 0.13 1.00 0.32 0.88 0.17 1.54 0.12
Sal
in various brain regions
Results expressed in ng/mg wet weight Significant differences from Saline treated animals as revealed by two tailed Dunnett tests: a p < 0.01; b p < 0.05.
Substantia Nigra Ventral Tegmentum
LD t.r SD
:D
:D
0.89 0.25 0.21 0.04 0.36 0.13 0.23 0.05 0.50 0.13 0.33 0.07 0.40 0.12 0.44 0.08
Sal
5 min 7.5
of neurotensin
s”D
!D
Effects
Hypothalamus
Septum
Frontal Cortex corpus Striatum Nucleus Accumbens Amygdala
Table 3
0.72 0.13 1.26 0.03 0.34b 0.17 0.76 0.10 1.04 0.18 1.04 0.27 0.72 0.24 1.23 0.16b
5 min 7.5
0.57 0.14 1.12 0.34 0.83 0.33 0.69 0.08 0.81 0.30 0.74 0.27 0.73 0.22 1.13” 0.35b
30.0
1.00 0.32 1.40 0.20 1.18 0.48 0.97 0.30 1.01 0.47 1.25 0.62 0.74 0.23 1.32 0.40
Sal
S-HT
0.87 0.15 1.27 0.20 1.11 0.23 0.92 0.23 1.51 0.38 0.99 0.14 0.82 0.17 1.65 0.33
30 min 7.5
0.98 0.20 1.46 0.13 2.2ob 0.71 1.11 0.12 1.04 0.50 1.31 0.27 0.73 0.19 1.07 0.27
30.0
EFFECTS OF NEUROTENSIN ON REGIONAL BRAIN CONCENTRATIONS
..
175
NT at both 5 (p < 0.05) and 30 (p < 0.01) min prior to decapitation. HVA/DA was similarly affected. Significant increases in 5-HIAAIS-HT were also found following the administration of 7.5 ~g 5 min prior to sacrifice (p c 0.05) and 30 pg 30 min prior to sacrifice (p < 0.01) (Table 3 and Figure). Amygdala: Both DOPAC/DA and HVAIDA were elevated 5 min after the injection of 30 pg NT (p’s < 0.01). No changes were observed for 5-HIAA/S-HT (Table 3). Septum : The only ratio significantly changed by NT admission in this region was HVA/DA at 5 min following the administration of 3Ok.g NT (p < 0.05) (Table 3). Hypothalamus : In the hypothalamus 3Ot.~g NT significantly elevated DOPAC/DA 30 min after injection (p < 0.05) while HVA/DA was increased 5 min after the administration of 3Op,g of the peptide, p < 0.01 (Table 3). Ventral Tegmentum : DOPACYDA was significantly increased 5 min following the administration of 3Op,g NT (p < 0.01). 5-HIAA/S-HT was significantly decreased by both doses of the peptide 5 min after administration, p < 0.05 (Table 3). Substantiu Nigra : In this region HVA/DA was significantly increased 5 min after the injection of 3Op,g NT, p < 0.05 (Table 3).
Discussion
Figure Effects of 0.0,7.5 or 3O.Opg neurotensin on metabolitekransmitter ratios in the nucleus accumbens. Panel A: 5 min following injection: Panel B: 30 min following injection. * p < 0.05; *+p< 0.01.
The results of this study suggest that the effects of NT on dopaminergic function are more pronounced and pervasive than previously suspected. As determined by changes in transmitter and/or metabolite concentrations, dopaminergic activity was increased to some extent in all regions studied except the frontal cortex and ventral tegmentum. In general the effects were dose related and were more pronounced 5 min following the administration of the peptide, where both 7.5 and 3Opg NT produced significant elevations in dopamine, (corpus striatum and amygdala) DOPAC (corpus striatum, nucleus accumbens, amygdala, septum and hypothalamus) and HVA (corpus striatum, nucleus accumbens, amygdala, septum, hypothalamus and substantia nigra) levels. These effects persisted in some but not all regions 30 min later, but only following the injection of the higher dose. It should be noted that, in contrast to
176 previously published data where the peptide was administered intracisternally (10, 12), the IVT injection of the peptide did not preferentially increase HVA as compared to DOPAC levels in most regions examined. Five min after injection both HVA and DOPAC increased in parallel, while at 30 min the levels of both metabolites remained elevated in the same regions with the exception of the septum (where DOPAC was no longer significantly increased) and the substantia nigra (where only HVA was elevated at both test periods.) The increase in dopaminergic transmission suggested by the above results were reflected by prominent increases in the ratios of each metabolite to dopamine. As can be seen in Table 3 the ratios of DOPAC/DA were enhanced in the frontal cortex, striatum, nucleus accumbens, amygdala, hypothalamus and ventral tegmentum. In the case of HVA/DA ratios, significant elevations were found in all regions examined. Since changes in metabolite to transmitter ratios are thought to reflect more reliably alterations in the rate of neurotransmitter utilization (14), the observed increases in both ratios provide additional evidence that NT increases DA turnover. It should be mentioned that increases in metabolite to DA ratios were found in regions constituting terminals of all main dopaminergic pathways; mesolimbic, mesostriatal and mesocortical. The fact that changes in DA turnover were found in this latter pathway indicates that the observed neurochemical effects of NT on DA metabolism are not mediated by an action of the peptide on inhibitory autoreceptors, since the frontal cortex purportedly lacks these receptors (13,15). Finally, it is interesting to note that NT increased the ratios of both metabolites to dopamine in the ventral tegmentum and substantia nigra, areas containing cells of origin of the main dopaminergic pathways. Again, as with changes in absolute concentrations, the effects of NT on metabolites to dopamine ratios were more evident at 5 than 30 min. This is intriguing since known behavioural effects of the peptides are most prominent at 30 min following IVT administration (19,20). This might suggest that there is no relationship between behavioural changes and alterations in dopaminergic activity. However, it could also be argued
NEUROPEPTIDES
that behavioural effects of the peptide are a lasting consequence of earlier modifications of dopaminergic function. It is tempting to speculate that the large and pervasive increases in metabolic concentrations as well as in the ratios of each metabolite to dopamine seen 5 min following injection are due to an inhibitory effect of the peptide on the efflux of acidic metabolites out of the brain. This effect has been encountered frequently, for example, following the administration of neuroleptics (21, 22). However, the results of the present study demonstrate that NT induces region specific increases in dopamine levels, and it has been shown that the peptide increases synthesis of dopamine (10, 1l), and promotes the release of dopamine in vitro (23), all of which militate against a non-specific inhibitory action of the peptide on active transport mechanisms. The effects of NT on serotonin metabolism were more variable and disparate (Table 2). NT produced a decrease in 5-HT in the nucleus accumbens with the 3Ol.~g dose at 30 min and in the hypothalamus following 7.5 pg, again 30 min after the administration of the peptide. The major metabolite of 5-HT, 5-HIAA, was increased in the nucleus accumbens 5 min following 7.5 pg, in the corpus striatum 30 min following 3Opg, and decreased in the hypothalamus 30 min following the administration of 7.5~g NT. When 5-HIAA ratios were examined, significant increases were found in the nucleus accumbens at both 5 and 30 min while this ratio was significantly decreased at 5 min in the ventral tegmentum. These results contrast with an earlier report where similar doses had no effect on either 5-HT or 5-HIAA concentrations in the same regions, though the effects were assessed only at 60 min following administration of the peptide (10). The physiological significance of the observed changes in serotoninergic indices remain to be elucidated; however, in view of the well known interaction between serotonin and dopamine (24,25), the possibility that some of the effects of NT on dopamine transmission might be mediated by an action of the peptide on serotoninergic processes merits further experi mental attention. Finally, the observed effects of the peptide on serotonin parameters demonstrate that the neurochemical effects of NT are not
EFFECTS OF NEUROTENSIN ON REGIONAL BRAIN CONCENTRATIONS
restricted to dopaminergic transmission. In accordance with this view, changes in norepinephrine and acetylcholine concentrations in various brain regions following administration of the peptide have been reported (l&26). In summary, the present results demonstrate that the effects of NT on dopaminergic transmission are more widespread than previously reported in that all major dopamine pathways are affected by the peptide. Also, the observed changes in the ratios of both DOPAC and HVA to DA suggest that NT increases the utilization of this transmitter. Finally, results indicate that NT influences serotoninergic transmission, although the observed effects are less pronounced and more region specific.
Acknowledgements This work was supported by the Medical Research Council of Canada, Development Grant DG-284.
References 1. Jolicoeur, F. B., Rioux, F. and St-Pierre, S. (1985). Neurotensin. Handbook of Neurochemistry 8: 93-114. 2. Nemeroff, C. B. (1986). The interaction of neurotensin with dopaminergic pathways in the central nervous system. Basic neurobiology and implications for the pathogenesis and treatment of schizoprenia. Psychoneuroendocrinology 11: 15-37. 3. Emson, P. C., Goedert, M., Horsfield, P., Rioux, F. and St-Pierre, S. (1982). The regional distribution and chromatographic characterization of neurotensin-like immunoreactivity in the rat central nervous system. J. Neurochem. 38: 992-looo. 4. Quirion, R., Gaudreau, P., St-Pierre, S., Rioux, F., and Pert, C. (1982). Autoradiographic distribution of [H3] neurotensin receptors in rat brain, Peptides 3: 757-763. 5. Hokfelt. T., Everitt, B. J., Theodorsson-Norheim, E., Goldstein, M. (1984). Occurrence of neurotensin-like immunoreactivity in sub-populations of hypothalamic, mesencephalic, and medullary catecholamine neurons. J. Comp. Neural. 222: 543-559. 6. Palacios, J. M., and Kuhar, M. J. (1981). Neurotensin receptors are located on dopamine-containing neurons in rat mid brain. Nature 294: 587-589. 7. Quirion, R. (1983). Interactions between neurotensin and dopamine in the brain: An overview. Peptides 4: 609-615.
177
8. Jolicoeur, F. B., DeMichelle, G., Barbeau, A., and St-Pierre, S. (1983). Neurotensin affects hyperactivity but not stereotypy induced by pre- and post synaptic dopaminergic stimulation. Neurosci. Biobehav. Revs. 7: 385 390. 9. Jolicoeur, F. B., Rives& R., St-Pierre, S., Gagne, A., and Dumais, M. (1985). The effects of neurotensin and [DTyrll]-N T on the hyperactivity induced by intra-accumbens administration of a potent dopamine receptor agonist. Neuropeptides 6: 143-156. 10. Widerlov. E.. Kilts, C. D., Mailman, R. B., Nemeroff, C. B., McCown, T. J., Prange, A. J. Jr. and Breese, G. R. (1982). Increase in dopamine metabolites in rat brain by neurotensin. J. Pharmac. Exp. Ther. 222: l-6. 11. Reches, A., Burke, R. I., Jiant, D. H., Wagner, H. R. and Fahn, S. (1983). Neurotensin interacts with dopaminergic neurons in rat brain. Peptides 4: 43-48. 12. Nemeroff. C. B.. Luttinger, D., Hernandez, D. E., Mailman, R. B., Mason, G. A., Davis, S. D., Widerlov, E.. Frye, G. D., Kilts, C. A., Beaumont, K., Breese, G. R. and Prange, A. J. Jr. Interactions of neurotensin with brain dopamine systems: biochemical and behavioral studies. J. Pharmac. Exp. Ther. 225: 337-345. 13. Bannon, M. J. and Roth, R. H. (1983). Pharmacology of mesocortical dopamine neurons. Pharmac. Rev. 35: 534-68. 14. Lavielle, S., Tassin. J-P., Thierry, A.-M., Blanc, G., Herve, D., Barthelamy, C., and Glowinski, J. (1978). Blockade by benzodiazepine of the selective high increase in dopamine turnover induced by stress in mesocortical dopaminergic neurons of the rat. Brain Res. 168: 585-594. 15. Sharp. T.. Zetterstrom. T. and Ungerstedt, U. (1986) An in vivo study of dopamine release and metabolism in rat brain regions using intracerebral dialysis. J. Neurochem. 47: 113-122. 16. Heffner, J., Hartman, P., and Seiden, L., (1980). A rapid method for dissection of the rat brain. Pharmac, Biochem, Behav. 13: 453-455. 17. Drumheller. A. L.. Gratton, N., Gagne, M., and Jolicoeur, F. B. (1986). Effects of neurotensin on regional concentrations of norepinephrine in rat brain. Brain Res. Bull. 16: 755-758. 18. St. Pierre, S. Lalonde, J. M., Gaudreau, M., Quirion, R., Regoli, D. and Rioux, F. (1981). Synthesis of peptides by the solid phase method: Neurotensin, fragments and analogues. J. Med. Chem. 24: 370-376. 19. Jolicoeur. F. B., Barbeau, A., Rioux, F.. Quirion, R. and St-Pierre, S. (1981). Differential neurobehavioral effects of neurotensin and structural analogues. Peptides 2: 171175. 20. Jolicoeur, F. B., Aube, C., St-Pierre, S., Rivest, R. and Gagne, M. (1984). Relationships between structure and duration of neurotensin’s central action: Emergence of long-acting analogues. Neuropeptides 4: 467-476. 21. Moleman. P., Bruinvels, J. and Van Valkenburg. C. F. M. (1978). Haloperidol inhibits the disappearance of acidic metabolites from the rat striatum. J. Pharm. Pharmac. 30: 583-585.
178 22. Westerink, B. H. C. (1985) Sequence and significance of dopamine metabolism in the rat brain. Neurochem. Int. 7: 221-227. 23. Okuma, Y., Fukuda, Y., Osumi, Y. (1983). Neurotensin potentiates the potassium induced release of endogenous dopamine from rat striatal slices. Eur. J. Pharmac. 93: 27-33. 24. Costall, B., Naylor, R. J., Marsden, C., Pycock, J. (1976). Serotoninergic modulation of the dopamine response from the nucleus accumbens 28: J. Pharm, Pharmac, 28: 523526.
NEUROPEPTIDES 25. Dray, A. L., Gonye, T. J., Oakley, N. R. and Tanner, T. (1976). Evidence for the existence of a raphe projection to the substantia nigra in rat. Brain Res. 113: 45-57. 26. Malthe-Sorenssen, D. Wood, P. L., Cheney, D. L. and Costa, E. (1978). Modulation of the turnover rate of acetylcholine in rat brain be intraventricular injections of thyrotropin-releasing hormone, somatostatin, neurotensin and angiotensin II. J. Neurochem. 31: 685-691.
0143-4179/90/0015-0169/$10.00
Effects of Neurotensin on Regional Brain Concentrations of Dopamine, Serotonin and their Main Metabolites A. D. DRUMHELLER”,
M. A. GAGNi*,
S. ST-PIERREt
and F. 6. JOLICOEUR*
*Department of Psychiatry, Faculty of Medicine, University of Sherbrooke, Sherbrooke, QuBbec, Canada, JlH 5N4 and tlNRS, 345 Hymus Blvd., Pointe Claire, QuBbec, Canada H9R lG6. (Reprint requests to FBJI
Abstract-The effects of neurotensin, 7.5 or 3Opg, on concentrations of DA, DOPAC, (HVA), serotonin 5-HT and 5-HIAA were measured in 8 regions of the rat brain either 5 or 30 min following intracerebroventricular administration. Regions examined included the frontal cortex, striatum, nucleus accumbens, amygdala, septum, hypothalamus, ventral tegmentum and substantia nigra. Results indicate that both doses of neurotensin significantly elevated concentrations of dopamine in the striatum and amygdala 5 min following injection. The effects of the peptide on DOPAC and HVA were more pervasive and enduring, with significant increases in metabolite levels occurring in both mesolimbic and nigrostriatal terminal regions. In order to assess effects on turnover of dopamine, the ratios of each metabolic to dopamine concentrations were examined. Results indicate that, while the DOPAC/DA ratio was elevated in many regions, the HVA/DA ratio was increased in all regions examined. The effects of neurotensin on serotoninergic parameters were less pervasive and more variable, with both increases and decreases in 5-HT and 5-HIAA concentrations being observed. The effects of the peptide on 5-HIAA/5-HT were limited to the nucleus accumbens, where this ratio was increased, and the ventral tegmentum, where 5-HIAA/5-HT was decreased. These findings reveal that the effects of the neurotensin on dopaminergic transmission are more widespread than previously reported in that all major dopamine pathways are affected by the peptide. Also, the observed changes in the ratios of both DOPAC and HVA to DA suggest that neurotensin enhances the turnover. of this transmitter.
Introduction Several lines of evidence suggest that the tridecapeptide, neurotensin (NT) interacts directly or indirectly with central dopaminergic systems (1, Date received 5 September 1989 Date accepted 5 September 1989
2). Neurotensin-like immunoreactivity is present in many areas containing dopamine (DA) cell bodies or terminals (3, 4). The peptide has been shown to be intraneuronally co-localized with DA in several discrete regions of the brain (5), and direct synaptic contact between NT and DA neurons has also been suggested (6,7). Behaviour-
169
170 ally, NT has been shown to decrease the hyperactivity induced by pre- and postsynaptic pharmacological stimulation of dopaminergic transmission (8, 9). Neurochemical studies have also supported a DA/NT interaction. Following DOPA decarboxylase inhibition, NT injected either intracisternally or intracerebroventricularly, increases DA synthesis as measured by DOPA accumulation in both mesolimbic and meostriatal terminals (10, 11). Some data suggest that the observed increase in DOPA is a result of a direct action of NT on presynaptic inhibitory DA receptors (11). The effects of centrally administered NT on regional levels of DA and its major metabolites, dihydrophenylacetic acid (DOPAC) and homovanillic acid (HVA) have also been studied and the results indicate that the neurochemical effects of the peptide are varied, depending on the dose administered and the time course examined. When administered intracisternally (IC), DA levels remain unchanged in the striatum, olfactory tubercles, nucleus accumbens and hypothalamus 60 min after the administration of 3, 30 or 100 pg NT (lo), but a later report from the same group indicated that an intermediate dose of 6Ol.~gNT increased levels of DA in the olfactory tubercle when measured 60 min after IC injection (12). On the other hand, the same authors reported a transient increase in striatal DA concentrations 30 min after administration of 30 u,g NT (10). The effects of the same doses of NT on regional DOPAC levels were also examined in the same regions. Significant elevations in this metabolite were found in the hypothalamus, striatum and olfactory tubercles, generally with the highest dose (lo), and in the nucleus accumbens with a lower (60 p_g) dose (12). The effects of the peptide on regional HVA levels appear to be more consistent. Dose dependent increases in this metabolite were found in all tissues examined (10, 12). Finally, when NT was injected intraventricularly (IVT), similar elevations in DOPAC and HVA concentrations were found with doses ranging from 3 to 1OOk.g in both striatum and nucleus accumbens (11). The purpose of the present study was to further investigate the effects of NT on dopamine metab-
NEURQPEPTIDES
olism in several projection areas of major dopaminergic pathways including the frontal cortex, amygdala, septum, nucleus accumbens, corpus striatum and hypothalamus. Furthermore, the effects of the peptide on dopamine metabolism within the regions containing the cells of origin of the dopamine projection systems, the substantia nigra and the ventral tegmentum, were also examined. The changes in dopamine metabolism were assessed following the IVT administration of both 7.5 and 3Okg of NT injected either 5 or 30 min prior to sacrifice. Finally, using the same experimental paradigm, the effects of the peptide on serotonin (5-HT) and its main metabolite, 5hydroxyindoleacetic acid (5-HIAA) were also assessed. Results are expressed as regional concentrations of DA, DOPAC, HVA, 5-HT and 5-HIAA in ng/mg wet weight, as well as the ratios DOPAC/ DA, HVAIDA and 5-HIAAIS-HT found in each region. It has been suggested that the use of metabolite/transmitter ratios provides a simple, reliable and non-invasive measure of neurotransmitter utilization (13, 14, 15).
Methods Animals
Male hooded rats obtained from Canadian Breeding Farm (St. Constant, Quebec) and weighing between 250 and 300g were used. Animals were housed in a temperature controlled room having a 12 hr light/dark cycle. Under pentobarbital anaesthesia, indwelling stainless steel cannulae were implanted into the left lateral cerebra-ventricle. Procedure
Following 48 hr of recovery, animals were divided into six groups (n = 8). Three groups of rats received either saline, 7.5 pg or 30 kg neurotensin in a volume of 20 kg1 0.9% NaCl, injected over a 30 set period by means of a 5Opl Hamilton syringe, and were sacrificed 5 min after injection. The other 3 groups received the same treatments, but were sacrificed 30 min after injection. Following a sharp blow to the head, brains were rapidly removed and placed on the dissection block on dry ice and regions excised according the method of Heffner
EFFECTS OF NEUROTENSIN ON REGIONAL BRAIN CONCENTRATIONS
et al. (16). The following regions were dissected: frontal cortex, hypothalamus, amygdala, nucleus accumbens, corpus striatum, substantia nigra, ventral tegmentum and septum. Regions were frozen at -80°C until analysis. All regions were homogenized in 0.1 M HC104 containing 400 p,l 1.OM sodium bisulphite. Homogenization volume for the cortex and amygdala was 1.Oml, and for the other regions, 400(~1. Brains were centrifuged at 15000rpm for 1.5min, the supernatants removed, filtered and 80 ~1 of each sample injected onto the column. Biochemical analysis
The separation and quantification of amines, indoleamines and their metabolites were carried out using a previously described method (17). Briefly, the chromatographic system (all components by Beckman Instruments) consisted of 2 pumps, a controller and an injector fitted with a 1OOpJ sample loop. An Ultrasphere ODS 5 l.~ reverse phase column (4mm x 15cm) was protected by a short precolumn. The column eluate was monitored by an LC 2A electrochemical detector (Bioanalytical Sytems) equipped with a glassy carbon electrode and operated at an applied potential of 0.65 V with reference to a silver/silver chloride electrode. The primary eluting solvent was a mixture of 0.1 M sodium acetate and 0.02M citric acid, pH 4.2 containing 0.3mM ethylenediamine-tetraacetic acid (EDTA) and 0.05mM sodium octysulfate. The secondary solvent was methanol. Amines were separated using a linear gradient of 08% methanol over 8 min, starting 4 min after injection. Twenty two minutes into the separation the % methanol was decreased to 0 over 2 min and the column re-equilibrated with starting buffer for 10 min prior to re-injection. All operations were performed at ambient temperature at a flow rate of 1.5mYmin. Injection volumes for standards and brain extracts remained constant at 8Ou.l. All solutions were filtered through 0.2l.~ nylon membranes (Ultipor, Chromatographic Specialties Co.) and thoroughly degassed prior to use. Quantitation of sample amine concentrations was performed by direct comparison of sample peak heights to the peak heights obtained follow-
171
ing the injection of known quantities of DA, DOPAC, HVA, 5-HIAA and 5-HT and converting to ng/mg wet weight. Chemicals
Neurotensin was synthesized by the solid phase synthesis technique according to a previously described procedure in our laboratory (18). DA, DOPAC, HVA, 5-HT and 5-HIAA were purchased from Sigma. Sodium acetate, citric acid, sodium bisulphite, HC104, EDTA and methanol were purchased from Fisher. Sodium octyl sulfate was purchased as a 40% solution from ICN. Distilled water was redistilled in glass and passed through a Norganic cartridge (Waters Associates) prior to use. Statistical analysis
For each time, region and substance measured, data were analyzed by means of individual oneway ANOVA’s. Levels of the main factor included treatments (0.9% NaCl, 7.5 and 3O.Op,g NT). Differences between saline controls and NT treated animals were assessed by means of Dunnett’s tests. In all cases a difference was considered significant if it had a probability of random occurrence of less than 5%. Results Effects of NT on regional concentrations of dopamine, DOPAC and HVA Dopamine. The effects of neurotensin on regional concentrations of dopamine are shown in Table 1. The levels of dopamine were significantly elevated in the corpus striatum and the amygdala by both 7.5 (p < 0.01) and 3OFg (p < 0.05) NT 5 min after injection. No significant effects of neurotensin were seen 30 min following administration in any region. DOPAC.
The effects of neurotensin on regional concentrations of DOPAC are shown in Table 1. Significant increases in DOPAC content were observed in the corpus striatum, nucleus accumbens, amygdala and hypothalamus 5 min following ICV injection of either 7.5 (p’s < 0.05) or 30.0 (p’s < 0.01) Fg of the peptide. In the septum, DOPAC
:D
:D
0.08 0.02 11.74” 2.56 7.50 1.49 0.66” 0.15 0.79 0.22 0.47 0.15 1.02 0.15 0.90 0.45
5 min 7.5 0.05 0.01 0.57b 2.07 7.29 1.72 0.61b 0.08 0.67 0.25 0.41 0.08 0.81 0.28 0.89 0.36
30.0 0.11 0.04 8.12 1.72 6.43 0.94 0.45 0.06 0.58 0.13 0.34 0.06 0.79 0.13 0.95 0.22
Sal 0.11 0.06 9.15 0.94 6.83 1.45 0.54 0.07 0.53 0.20 0.37 0.07 0.75 0.20 1.37 0.50
30 min 7.5 0.08 0.04 9.27 1.78 7.20 3.45 0.55 0.12 0.60 0.17 0.36 0.12 0.97 0.54 1.14 0.39
30.0 0.07 0.03 1.66 0.21 2.14 0.47 0.11 0.03 0.28 0.07 0.13 0.03 0.39 0.09 0.42 0.11
Sal 0.06 0.02 2.37 0.29 3.10b 0.49 0.16b 0.03 0.27 0.09 0.22b 0.07 0.41 0.09 0.38 0.18
5 min 7.5 0.09 0.02 2.83” 0.84 3.50” 0.75 0.29” 0.11 0.47” 0.17 0.44” 0.05 0.47 0.15 0.54 0.18
30.0 0.09 0.06 1.62 0.44 2.52 1.12 0.10 0.03 0.28 0.07 0.17 0.08 0.43 0.23 0.44 0.15
Sal
DOPAC
0.05 0.01 1.54 0.45 2.19 0.34 0.14 0.03 0.21 0.09 0.13 0.03 0.37 0.19 0.38 0.10
30 min 7.5 0.09 0.03 2.24b 0.78 4.07b 1.19 0.16 0.06 0.34 0.10 0.28” 0.06 0.52 0.27 0.40 0.12
30.0
of Dopamine, DOPAC and HVA
Results expressed in ng/mg wet weight Significant differences from Saline treated animals as revealed by two tailed Dunnett tests: a p < 0.01; b p < 0.05.
Substantia Nigra Ventral Tegmentum
!D u SD
:D
:D
!D
0.07 0.03 8.25 1.69 6.18 1.29 0.49 0.06 0.62 0.17 0.38 0.06 0.91 0.17 1.97 0.21
Sal
DOPAMZNE
of neurotensin on regional brain concentrations
ED
Effects
Hypothalamus
Septum
Striatum Nucleus Accumbens Amygdala
COlpS
Frontal Cortex
Table 1
0.10 0.02 0.82 0.06 0.91 0.11 0.09 0.03 0.21 0.02 0.05 0.02 0.34 0.09 0.37 0.05
Sal 0.11 0.03 1.23” 0.14 1.15b 0.33 0.12b 0.02 0.19 0.06 0.07 0.01 OMb 0.33 0.33 0.25
5 min 7.5
0.12 0.04 1.21” 0.41 1.37” 0.22 0.18” 0.05 0.51” 0.27 0.15” 0.07 0.83” 0.38 0.43 0.13
30.0
30 min 7.5 0.09 0.03 0.92 0.26 0.83 0.12 0.10 0.05 0.12 0.03 0.06 0.02 0.16 0.07 0.34 0.26
Sal 0.08 0.02 0.77 0.21 0.87 0.22 0.07 0.04 0.18 0.05 0.07 0.02 0.28 0.09 0.40 0.24
HVA
0.07 0.02 lLKrb 0.34 1.26” 0.23 0.11 0.03 0.26b 0.05 0.08 0.03 0.45 0.08 0.45 0.08
30.0
EFFECTS OF NEUROTENSIN ON REGIONAL BRAIN CONCENTRATIONS
levels were significantly increased at 5 min by the higher dose of NT (p < 0.01). Thirty min following administration of 3Ol.~gNT, DOPAC levels were significantly increased in the corpus striatum, nucleus accumbens (p’s < 0.05) and hypothalamus (p < 0.01). HVA.
The effects of neurotensin on regional concentrations of HVA are shown in Table 1. Significant increases in HVA levels were found in the corpus striatum 5 min after the administration of each dose of the peptide (p’s < 0.01). The administration of either 7.5 (p’s < 0.05) or 30.0 (p’s < 0.01) pg NT 5 min prior to decapitation also resulted in elevated HVA concentrations in the nucleus accumbens, amygdala and substantia nigra. In the septum and hypothalamus, only the higher dose of the peptide increased HVA levels at this time period (p < 0.01). HVA levels were still elevated 30 min after the injection of 30.0 but not 7.5 u,g of NT in the nucleus accumbens (p < O.Ol), corpus striatum, septum and substantia nigra (p’s < 0.05).
nucleus accumbens (p < 0.05) and in the hypothalamus following the administration of 7.5 pg of the peptide. These results are shown in Table 2. SHZAA.
Tissue levels of 5-HIAA were significantly increased 5 min after the injection of 7.5 Fg NT in the nucleus accumbens (p c 0.05). When measured 30 min following injection, SHIAA levels were increased in the corpus striatum with the higher dose of the peptide (p < 0.05) and was decreased in the hypothalamus with the lower dose tested (p < 0.01) (Table 2).
Effects of NT on regional concentrations of 5-HT and 5-HIAA 5-HT. The concentration
of 5-HT was decreased 30 min following the injection of 3Ocl.gNT in the Table 2
Effects of neurotensin
173
Effects of NT on metabolitelneurotransmitter
as well as the HVA/DA ratios were significantly increased in the frontal cortex following 3O~g NT both 5 and 30 min after injection (p’s c 0.01) (Table 3). No changes were observed in the ratio of 5-HIAA/5HT. Corpus striatum : The DOPAC/DA ratio was significantly increased by the 3Okg dose 30 min following administration (p < 0.05). HVA/DA was increased 5 min after the injection of 30 kg NT (p < 0.05) (Table 3). No changes were observed in the ratio 5-HIAA-5-HT. Nucleus accumbens : The DOPACYDA ratio was significantly increased in this region following the administration of 3Opg
on regional brain concentrations
of serotonin and 5-HIAA
Serotonin Sal
Frontal Cortex Corpus Striatum
ED
0.40 0.09 0.55
5 min 7.5
30.0
Sal
.5-HIAA 30 min 7.5
0.47
0.38
0.26
0.10 0.51 0.06 0.66
0.08
0.11
0.42
0.40
0.33 0.04 0.46
0.09 0.70
0.15 0.52
0.06 0.67
Nucleus
+
0.14 0.64
Accumbens
SD
0.17
0.15
0.22
0.13
0.23
0.50 0.12 0.30 0.05 0.69 0.15 0.87 0.30 0.73 0.26
0.56 0.15 0.35 0.09 0.74 0.15 0.91 0.31 0.70 0.27
0.46 0.08 0.58 0.21 0.65 0.16 1.22 0.36 0.99 0.30
0.48 0.09 0.34 0.15 0.61 0.21 1.10 0.36 0.93 0.14
0.42 0.17 0.25 0.10 0.38b 0.18 0.79 0.16 0.90 0.48
ED
Amygdala :D Septum Hypothalamus Substantia Nigra Ventral Tegmentum
:D u SD ;D s”D
ratios
Frontal cortex. The DOPAUDA
30.0
Sal
5 min 7.5
0.29 0.05
0.23 0.11
0.41 0.08 0.31” 0.14 0.44 0.07 0.41 0.12 0.55 0.15 0.95 0.36 0.88 0.14
30.0
Sal
30 min 7.5
30.0
0.32
0.19
0.07
0.09
0.21 0.10
0.28 0.02
0.28 0.05
0.56
0.59
0.42
0.45
0.57
0.61b
0.17 0.62 0.20 0.41 0.12 0.42 0.09 0.68 0.22 0.77 0.33 1.15 0.34
0.18 0.86b 0.12 0.44 0.11 0.34 0.06 0.74 0.12 0.84 0.20 1.01 0.24
0.10 0.61 0.20 0.33 0.06 0.54 0.31 0.47 0.11 0.83 0.17 1.03 0.21
0.15
0.09
0.08
0.59
0.70
0.65
0.17 0.47 0.06 0.32 0.08 0.73 0.19 0.76 0.17 1.23 0.33
0.17 0.37 0.10 0.37 0.06 0.40a 0.08 0.63 0.09 1.35 0.29
0.21 0.49 0.12 0.38 0.09 0.73 0.22 0.64 0.20 0.96 0.16
Results expressed in ng/mg wet weight Significant differences from Saline treated animals as revealed by two tailed Dunnett tests: a p < 0.01; b p < 0.05.
ED
s”D
0.73 0.19 0.21 0.04 0.43 0.11 0.24 0.07 0.36 0.10 0.55 0.29 0.40 0.12 0.40 0.07
1.7LGb 0.66 0.26 0.08 0.53b 0.18 0.49b 0.17 0.66 0.17 0.56 0.23 0.50 0.08 0.63” 0.12
30.0 0.82 0.21 0.19 0.02 0.42 0.13 0.26 0.13 0.47 0.12 0.48 0.19 0.52 0.21 0.41 0.05
Sal
HVAIDA
0.61 0.17 0.17 0.04 0.33 0.07 0.27 0.08 0.39 0.17 0.37 0.14 0.41 0.13 0.31 0.12
30 min 7.5
on ratios of metabolite
1.62” 0.47 0.24b 0.05 0.61” 0.18 0.28 0.06 0.57 0.10 0.92b 0.41 0.73 0.56 0.34 0.06
30.0 1.32 0.35 0.10 0.02 0.14 0.02 0.15 0.03 0.33 0.04 0.15 0.05 0.36 0.06 0.36 0.09
Sal
to transmitter
1.26 0.30 0.11 0.02 0.15 0.03 0.19 0.05 0.28 0.12 0.16 0.07 0.39 0.12 0.74 0.39
5 min 7.5 2.69” 0.75 0.14b 0.03 0.19” 0.03 0.29” 0.06 0.82b 0.40 0.31b 0.11 0.54b 0.25 1.29b 0.85
30.0 0.80 0.25 0.10 0.02 0.15 0.04 0.15 0.06 0.40 0.40 0.21 0.06 0.34 0.05 0.35 0.09
Sal
5-HIAA
concentrations
0.92 0.28 0.11 0.03 0.13 0.07 0.18 0.05 0.27 0.05 0.15 0.04 0.26 0.08 0.25 0.03
30 min 7.5 1.83” 0.50 0.12 0.03 0.20 0.07 0.18 0.06 0.40 0.07 0.25 0.14 0.42 0.18 0.46 0.18
30.0 0.75 0.21 1.04 0.29 0.97 0.28 0.80 0.20 1.29 0.13 1.00 0.32 0.88 0.17 1.54 0.12
Sal
in various brain regions
Results expressed in ng/mg wet weight Significant differences from Saline treated animals as revealed by two tailed Dunnett tests: a p < 0.01; b p < 0.05.
Substantia Nigra Ventral Tegmentum
LD t.r SD
:D
:D
0.89 0.25 0.21 0.04 0.36 0.13 0.23 0.05 0.50 0.13 0.33 0.07 0.40 0.12 0.44 0.08
Sal
5 min 7.5
of neurotensin
s”D
!D
Effects
Hypothalamus
Septum
Frontal Cortex corpus Striatum Nucleus Accumbens Amygdala
Table 3
0.72 0.13 1.26 0.03 0.34b 0.17 0.76 0.10 1.04 0.18 1.04 0.27 0.72 0.24 1.23 0.16b
5 min 7.5
0.57 0.14 1.12 0.34 0.83 0.33 0.69 0.08 0.81 0.30 0.74 0.27 0.73 0.22 1.13” 0.35b
30.0
1.00 0.32 1.40 0.20 1.18 0.48 0.97 0.30 1.01 0.47 1.25 0.62 0.74 0.23 1.32 0.40
Sal
S-HT
0.87 0.15 1.27 0.20 1.11 0.23 0.92 0.23 1.51 0.38 0.99 0.14 0.82 0.17 1.65 0.33
30 min 7.5
0.98 0.20 1.46 0.13 2.2ob 0.71 1.11 0.12 1.04 0.50 1.31 0.27 0.73 0.19 1.07 0.27
30.0
EFFECTS OF NEUROTENSIN ON REGIONAL BRAIN CONCENTRATIONS
..
175
NT at both 5 (p < 0.05) and 30 (p < 0.01) min prior to decapitation. HVA/DA was similarly affected. Significant increases in 5-HIAAIS-HT were also found following the administration of 7.5 ~g 5 min prior to sacrifice (p c 0.05) and 30 pg 30 min prior to sacrifice (p < 0.01) (Table 3 and Figure). Amygdala: Both DOPAC/DA and HVAIDA were elevated 5 min after the injection of 30 pg NT (p’s < 0.01). No changes were observed for 5-HIAA/S-HT (Table 3). Septum : The only ratio significantly changed by NT admission in this region was HVA/DA at 5 min following the administration of 3Ok.g NT (p < 0.05) (Table 3). Hypothalamus : In the hypothalamus 3Ot.~g NT significantly elevated DOPAC/DA 30 min after injection (p < 0.05) while HVA/DA was increased 5 min after the administration of 3Op,g of the peptide, p < 0.01 (Table 3). Ventral Tegmentum : DOPACYDA was significantly increased 5 min following the administration of 3Op,g NT (p < 0.01). 5-HIAA/S-HT was significantly decreased by both doses of the peptide 5 min after administration, p < 0.05 (Table 3). Substantiu Nigra : In this region HVA/DA was significantly increased 5 min after the injection of 3Op,g NT, p < 0.05 (Table 3).
Discussion
Figure Effects of 0.0,7.5 or 3O.Opg neurotensin on metabolitekransmitter ratios in the nucleus accumbens. Panel A: 5 min following injection: Panel B: 30 min following injection. * p < 0.05; *+p< 0.01.
The results of this study suggest that the effects of NT on dopaminergic function are more pronounced and pervasive than previously suspected. As determined by changes in transmitter and/or metabolite concentrations, dopaminergic activity was increased to some extent in all regions studied except the frontal cortex and ventral tegmentum. In general the effects were dose related and were more pronounced 5 min following the administration of the peptide, where both 7.5 and 3Opg NT produced significant elevations in dopamine, (corpus striatum and amygdala) DOPAC (corpus striatum, nucleus accumbens, amygdala, septum and hypothalamus) and HVA (corpus striatum, nucleus accumbens, amygdala, septum, hypothalamus and substantia nigra) levels. These effects persisted in some but not all regions 30 min later, but only following the injection of the higher dose. It should be noted that, in contrast to
176 previously published data where the peptide was administered intracisternally (10, 12), the IVT injection of the peptide did not preferentially increase HVA as compared to DOPAC levels in most regions examined. Five min after injection both HVA and DOPAC increased in parallel, while at 30 min the levels of both metabolites remained elevated in the same regions with the exception of the septum (where DOPAC was no longer significantly increased) and the substantia nigra (where only HVA was elevated at both test periods.) The increase in dopaminergic transmission suggested by the above results were reflected by prominent increases in the ratios of each metabolite to dopamine. As can be seen in Table 3 the ratios of DOPAC/DA were enhanced in the frontal cortex, striatum, nucleus accumbens, amygdala, hypothalamus and ventral tegmentum. In the case of HVA/DA ratios, significant elevations were found in all regions examined. Since changes in metabolite to transmitter ratios are thought to reflect more reliably alterations in the rate of neurotransmitter utilization (14), the observed increases in both ratios provide additional evidence that NT increases DA turnover. It should be mentioned that increases in metabolite to DA ratios were found in regions constituting terminals of all main dopaminergic pathways; mesolimbic, mesostriatal and mesocortical. The fact that changes in DA turnover were found in this latter pathway indicates that the observed neurochemical effects of NT on DA metabolism are not mediated by an action of the peptide on inhibitory autoreceptors, since the frontal cortex purportedly lacks these receptors (13,15). Finally, it is interesting to note that NT increased the ratios of both metabolites to dopamine in the ventral tegmentum and substantia nigra, areas containing cells of origin of the main dopaminergic pathways. Again, as with changes in absolute concentrations, the effects of NT on metabolites to dopamine ratios were more evident at 5 than 30 min. This is intriguing since known behavioural effects of the peptides are most prominent at 30 min following IVT administration (19,20). This might suggest that there is no relationship between behavioural changes and alterations in dopaminergic activity. However, it could also be argued
NEUROPEPTIDES
that behavioural effects of the peptide are a lasting consequence of earlier modifications of dopaminergic function. It is tempting to speculate that the large and pervasive increases in metabolic concentrations as well as in the ratios of each metabolite to dopamine seen 5 min following injection are due to an inhibitory effect of the peptide on the efflux of acidic metabolites out of the brain. This effect has been encountered frequently, for example, following the administration of neuroleptics (21, 22). However, the results of the present study demonstrate that NT induces region specific increases in dopamine levels, and it has been shown that the peptide increases synthesis of dopamine (10, 1l), and promotes the release of dopamine in vitro (23), all of which militate against a non-specific inhibitory action of the peptide on active transport mechanisms. The effects of NT on serotonin metabolism were more variable and disparate (Table 2). NT produced a decrease in 5-HT in the nucleus accumbens with the 3Ol.~g dose at 30 min and in the hypothalamus following 7.5 pg, again 30 min after the administration of the peptide. The major metabolite of 5-HT, 5-HIAA, was increased in the nucleus accumbens 5 min following 7.5 pg, in the corpus striatum 30 min following 3Opg, and decreased in the hypothalamus 30 min following the administration of 7.5~g NT. When 5-HIAA ratios were examined, significant increases were found in the nucleus accumbens at both 5 and 30 min while this ratio was significantly decreased at 5 min in the ventral tegmentum. These results contrast with an earlier report where similar doses had no effect on either 5-HT or 5-HIAA concentrations in the same regions, though the effects were assessed only at 60 min following administration of the peptide (10). The physiological significance of the observed changes in serotoninergic indices remain to be elucidated; however, in view of the well known interaction between serotonin and dopamine (24,25), the possibility that some of the effects of NT on dopamine transmission might be mediated by an action of the peptide on serotoninergic processes merits further experi mental attention. Finally, the observed effects of the peptide on serotonin parameters demonstrate that the neurochemical effects of NT are not
EFFECTS OF NEUROTENSIN ON REGIONAL BRAIN CONCENTRATIONS
restricted to dopaminergic transmission. In accordance with this view, changes in norepinephrine and acetylcholine concentrations in various brain regions following administration of the peptide have been reported (l&26). In summary, the present results demonstrate that the effects of NT on dopaminergic transmission are more widespread than previously reported in that all major dopamine pathways are affected by the peptide. Also, the observed changes in the ratios of both DOPAC and HVA to DA suggest that NT increases the utilization of this transmitter. Finally, results indicate that NT influences serotoninergic transmission, although the observed effects are less pronounced and more region specific.
Acknowledgements This work was supported by the Medical Research Council of Canada, Development Grant DG-284.
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