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Acute nicotine injections induce c-fos mostly in non-dopaminergic neurons of the midbrain of the rat
162
Molecular Brain Research, 20 (1993) 162-170 ~ 1993 Elsevier Science Publishers B.V. All rights reserved 0169-328x/93/$06.00
BRESM 70654
Acute nicotine injections induce c-fos mostly in non-dopaminergic neurons of the midbrain of the rat Ying Pang, Hideo Kiba and A. Jayaraman Department of Neurology. LSU School of Medicine, New Orleans, LA 70112 (USA) (Accepted 20 April 1993)
Key words: Protooncogene; Reinforcement: Mecamylamine; Fos; Accessory optic system: Ventral tegmental area: Dopamine; Nicotine
Induction of c-los gene is an immediate and early response in the cascade of molecular events that ultimately lead to long-term alterations in gene expression in neurons. The psychomotor stimulant and positive reinforcing effects of nicotine have been speculated to be mediated by the dopaminergic neurons of the ventral tegmental area (VTA). To identify the precise subsets of VTA neurons of the rat that mediate the acute nicotinergic effects, the pattern of expression of c-los gene was mapped using immunocytochemical methods. Acute nicotine injections resulted in prominent Fos-like immunoreactivity (-LI) in the medial terminal nucleus of the accessory optic system, the interpeduncular nucleus, and in the caudal linear subnucleus of VTA. The neurons of other VTA subnuclei, viz., the rostral linear, paranigralis, nucleus parabrachialis pigmentosus, and nucleus interfascicularis or the substantia nigra pars compacta did not contain any cells with Fos-LI. Mecamylamine abolished Fos-LI in most of the VTA neurons. These results suggest that acute nicotine injections induce c-los expression mostly in non-dopaminergic neurons of the ventral tegmental area of the rat and that nicotine induces c-los most intensely in the interpeduncular nucleus, the superior colliculus, and several other subnuclei of the accessory optic system.
INTRODUCTION
VTA DA soma and the terminals in the nucleus accumbens4,5,6,14,18,22.2529,4o.
Nicotine is a psychomotor stimulant and a positive reinforcer ~,15.20. The precise CNS sites and the mechanisms with which nicotine enhances the positive effects and leads to repeated self-administrative behavior are poorly understood. Compelling evidence from several laboratories has lead to the establishment of a working hypothesis that the mesolimbic dopaminergic (DA) neurons of the ventral tegmental area (VTA) and their projections play a major role in mechanisms underlying emotion, motivation, reward and reinforcement behavior 1t'24'42. Several studies indeed suggest that nicotine has an excitatory effect on the midbrain dopaminergic neurons, nicotine stimulates the A10 dopaminergic neurons of the VTA more potently and selectively than the A9 cells in the substantia nigra pars compacta (SNpc), and these stimulatory effects appear to be mediated by the nicotinic receptors localized on the
The mesencephalic DA neurons may be divided into many subdivisions and these individual subnuclei have afferent and efferent projections 12'1'~'3°'33'36 and neurochemical organization that is specific to each of the subnuclei 23"34'4t. It is possible that each of the subnuclei may contribute to different aspects of the 'reward" experience. As a crucial step towards understanding the neurochemical and molecular mechanisms underlying the interactions between nicotine and the midbrain DA neurons, it is important to identify the specific subnuclei of the ventral tegmental neurons that mediate the nicotinergic effects. In order to achieve this goal, experiments were conducted to define the distribution of the VTA cells expressing the protooncogene c-los after acute injections of nicotine. The c-los gene encodes a nuclear protein transcription modulator (Fos) that exhibits DNA binding in
Correspondence." A. Jayaraman, Department of Neurology, LSU School of Medicine, 1542 Tulane Ave., New Orleans, LA 70112, USA. Fax: (1) (504) 568-4084.
163 vitro. T h e c-los g e n e is o n e o f several i m m e d i a t e e a r l y g e n e s d i s t i n g u i s h e d by t h e i r r a p i d a n d t r a n s i e n t t r a n s c r i p t i o n a l activation. T h e g e n e c-fos is i n d u c e d by a
variety of e x t r a c e l l u l a r stimuli. T h e s e i n c l u d e electrically a n d n e u r o c h e m i c a l l y i n d u c e d seizures, electrical, physiological, n o c i c e p t i v e a n d m e t a b o l i c stimulation. In
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Fig. 1. Fos-LI was noted in neurons of the supramamillary nucleus (SUM), retrorubral (RRF) and peripeduncular areas, lateral and dorsal terminal nuclei even in saline and drug-naive rats (A,B) as well as in the saline-injected 'control' rats (C,D). Note the absence of Fos-LI in the superior colliculus and the SN complex.
164 Several studies have shown that induction of c-fos can be used as a marker, with single-cell specificity, to observe the temporal and spatial involvement of cer-
PC12 cells, c-los is induced by membrane depolarization and stimulation of nicotinic acetylcholine receptors (reviewed in ref. 9).
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Fig. 2. After an injection of 0.4 mg of s.c. nicotine, neurons of the medial terminal nucleus of the accessory optic system (MT) (A) and the interpeduncular nucleus (IP) (B,C,D) contained cells labeled intensely with Fos-LI. Note that some cells in the lateral terminal nucleus (A) and some in the dorsal terminal nucleus (C) are labeled with Fos-LI. A m o n g the many subdivisions of V T A the most prominent labeling was noted in caudal liner nucleus (CLI) (C,D), but Fos-LI was not seen in the interfascicular, paranigralis nuclei and nucleus parabrachialis pigmentosus of V T A (A,B). The superior collieulus and the periaqueductal gray were densely labeled with Fos-LI ( A - D ) in all experiments.
165 tain subsets of neurons following seizures. Using similar principles, cocaine- and amphetamine-responsive cells have been mapped in the striatum ~7. MATERIALS AND METHODS Experiments were conducted in several groups of male SpragueDawley rats (250-350 g). One group of control rats (n = 3) was saline
and drug naive whereas the second group of control rats (n = 4) was nicotine naive but received saline 1.0 ml alone. The experimental group of rats received subcutaneous injections of 0.1 mg (n = 3), 0.4 mg (n = 5), 0.8 mg (n = 4), 1.0 mg (n = 5) and 1.4 mg/kg (n = 3) of nicotine (Sigma, St. Louis, MO) in 0.9% NaCI (pH 7.0+0.2). The selection of different doses of nicotine adopted in our study was based on nicotine doses used by London et al. 26'27'28 in their studies of nicotine-induced local cerebral glucose utilization patterns. Another group (n = 3) received 1 mg/kg of mecamylamine (Sigma, St. Louis, MO) 30 min prior to 0.4 mg/kg of nicotine injections. The
Fig. 3. Photomicrographs show promment Fos-immunoreactive neurons in MT (A), interpeduncular nucleus (C) caudal linear subnucleus of VTA (D) and the superior colliculus (F). Note that the VTA subnuclei and substantia nigra do not contain any neurons with Fos-LI. Experiments using in situ hybridization methods with an oligonucleotide probe directed against rat cDNA for TH clearly show prominent hybridization signal in the VTA and substantia nigra pars compacta (SNC) (B), but not in MT. Nicotine-induced c-fos expression in VTA was blocked by mecamylamine (E) administered 30 min prior to nicotine injections.
166 mecamylamine dose was selected on the basis of the the dose of the drug adopted in the studies of Rosecrans et al. 32. The majority of the animals were sacrificed 2 h after nicotine injections, but one group of rats (n = 3) received 0.4 mg/kg of nicotine and were sacrificed 4 h after nicotine injections. The control and treated rats were anesthetized with sodium pentobarbital (25 mg/kg., i.p.,) and were perfused intracardially with a solution containing 4% paraformaldehyde in sodium phosphate pH 7.4. The brains were removed and postfixed in perfusate for about 1 h. The brains were blocked and stored overnight in sucrose solution. Subsequently the brains were cut into 40/.~ thick sections. The sections were then processed for the standard avidin-biotin immunohistochemical procedure 19 using peroxidase Vectastain Elite ABC Kit (Vector laboratories, Burlingame, CA) and by following the protocol suggested by the manufacturer. For detecting Fos the sections were incubated in 1:500 to 1:1000 concentration of a polyclonal antibody for Fos (PC05, Oncogene Science, Uniondale, NY). Control sections were incubated in the presence of Fos peptides (PC09, PC10, Oncogene Science, Uniondale, NY) to which the antibodies were raised. In few cases, adjacent sections were processed for Fos-like immunoreactivity (Fos-LI) and for localizing neurons that contain mRNA for rat tyrosine hydroxylase with in situ hybridization methods. The oligoprobes used in these experiments were directed against the nucleotide sequence of 1441-1485 of the rat tyrosine hydroxylase cDNA2. The sections were studied with darkfield and brightfield microscopy, and projection drawings were made using camera lucida. The cytoarchitectonic details of various nuclei of the midbrain and especially VTA were defined in adjacent sections counterstained with cresyl violet. The atlas of Paxinos and Watson 31 was used to identify various mesencephalic nuclei. For quantitative analyses, the number of Fos positive cells was ascertained by manually counting neurons with Fos-Ll within a 10 x 10 ~2 area of the superfical layers of the superior colliculus, MT and the interpeduncular nucleus under 40 x magnification. RESULTS
Distribution pattern of cells with Fos-LI in saline and drug naiL,e rats F o s - L I is n o t e d in m a n y n e u r o n s of t h e m i d b r a i n even in saline a n d d r u g naive rats. M a j o r i t y of t h e s e n e u r o n s are seen in t h e s u p r a m a m i l l a r y nucleus. F e w n e u r o n s with F o s - L I are also n o t e d in t h e p e r i p e d u n c u lar region, b u t n o n e are seen in t h e i n t e r p e d u n c u l a r nucleus, s u p e r i o r colliculus, V T A o r the s u b s t a n t i a nigra c o m p l e x (Fig. 1A, B).
Distribution pattern of cells with Fos-LI in saline-treated rats F o s - L I is n o t e d in m a n y n e u r o n s of t h e m i d b r a i n in saline t r e a t e d animals. Cells of the s u p r a m a m i l l a r y nucleus are p r o m i n e n t l y l a b l e d with F o s - L I . N e u r o n s l a b e l e d with F o s - L I a r e also n o t i c e d in an a r e a l a t e r a l to the s u p r a m a m i l l a r y nucleus and e x t e n d i n g in an a r e a ventral to m e d i a l lemniscus a n d v e n t r o m e d i a l to m e d i a l g e n i c u l a t e nucleus in t h e p e r i p e d u n c u l a r region. A m o n g the various subnuclei o f the V T A , few cells l a b e l e d with F o s - L I are n o t e d in the rostral l i n e a r
nucleus, i n t e r f a s i c u l a r nucleus a n d c a u d a l l i n e a r nucleus, but cells with F o s - L I are a b s e n t in the nucleus parabrachialis pigmentosus, paranigratis, interpeduncular nucleus, superficial layers o f the s u p e r i o r colliculus, a n d m e d i a l t e r m i n a l nucleus of the optic tract (Fig.
1C,D). Distribution pattern of Fos-LI in nicotine-injected rats Injections of O.1 mg / kg of nicotine A f t e r 0.1 m g / k g o f s.c., m a n y cells in the s u p r a m a m i l l a r y nucleus a n d the p e r i p e d u n c u l a r regions are l a b e l e d with F o s - L I . In a d d i t i o n significant n u m b e r o f F o s - r e a c t i v e cells a r e also n o t e d in the superficial layers o f the s u p e r i o r colliculus, the m e d i a l t e r m i n a l nucleus o f the accessory optic system (MT), a n d the i n t e r p e d u n c u l a r nucleus (not shown in figures).
Injections of 0.4 mg / kg of nicotine T h e p a t t e r n of d i s t r i b u t i o n o f n e u r o n s with F o s - L I in V T A b e c o m e m o r e clearly d e f i n e d as the n u m b e r of F o s - r e a c t i v e cells i n c r e a s e d d r a m a t i c a l l y w h e n t h e dose of nicotine injected is i n c r e a s e d to 0.4 m g / k g s.c. A f t e r 0.4 m g / k g o f s.c nicotine, at the level of t h e p o s t e r i o r h y p o t h a l a m u s , t h e s u p r a m a m i l l a r y nucleus c o n t a i n e d significant n u m b e r o f p r o m i n e n t l y l a b e l e d cells with F o s - L I . A t the rostral most level o f V T A , the m o s t i n t e n s e l a b e l i n g is n o t e d in M T (Figs. 2 A a n d 3A). T h e M T e x t e n d s f r o m an a r e a m e d i a l to t h e m e d i a l - m o s t a r e a of s u b s t a n t i a nigra p a r s c o m p a c t a ( S N p c ) a n d spans d o r s o l a t e r a l l y into a region v e n t r a l to m e d i a l l e m n i s c u s a n d p a r a l l e l to the d o p a m i n e r g i c n e u r o n s of SNpc. O n t h e basis of n e u r a l connectivity p a t t e r n M T has b e e n i d e n t i f i e d to have v e n t r a l a n d d o r s a l subdivisions. L a b e l e d cells a r e seen t h r o u g h o u t the r o s t r o c a u dal e x t e n t of b o t h ventral a n d d o r s a l subdivisions of M T (Figs. 2 A a n d 3A). A t rostral V T A levels, cells l a b e l e d with F o s - L I are few in the nucleus interfascicularis, a n d p a r a n i g r a l i s , but n o n e a r e n o t e d in n e u r o n s o f nucleus p a r a b r a c h i a l i s p i g m e n t o s u s , a n d s u b s t a n t i a nigra p a r s c o m p a c t a ( S N p c ) which c o n t a i n m R N A for tyrosine hydroxylase (Figs. 2A, B a n d 3A,B). A t m i d a n d c a u d a l V T A levels, cells with F o s - L I are l a b e l e d intensely in the i n t e r p e d u n c u l a r nucleus. T h e labeling p a t t e r n is n o t e d t h r o u g h o u t t h e r o s t r o c a u d a l extent o f this nucleus, b u t t h e rostral a n d apical divisions of t h e nucleus c o n t a i n e d m o r e cells l a b e l e d with F o s - L I t h a n the i n t e r m e d i a t e a n d l a t e r a l divisions (Figs. 2B,D a n d 3C). A m o n g the m a n y subdivisions o f V T A the most p r o m i n e n t labeling is n o t e d in c a u d a l l i n e a r nucleus ( C L I ) especially at the m i d a n d c a u d a l levels of the nucleus (Figs. 2C,D and 3D). T h r o u g h o u t the entire
167 TABLE I
Two hours post injections Saline and nicotine-naive (n = 3) Saline (n = 4) 0.1 m g / k g nicotine s.c. (n = 3) 0.4 m g / k g nicotine s.c. (n = 5) 0.8 m g / k g nicotine s.c. (n = 4) 1.0 m g / k g nicotine s.c. (n = 5) 1.4 m g / k g nicotine s.c. (n = 3) Mecamylamine + nicotine (1.0 r n g / k g + 0.4 m g / k g ) (n = 3) Four hours post injections 0.4 m g / k g nicotine s.c. (n = 3)
MT (mean 4- S.D.)
IP (mean 4- S.D.)
SC (mean 4- S.D.)
0.00 +_0.00 0.00 :t: 0.00 5.00 _+1.00 23.67 4. 4.32 24.50 _+2.08 25.33 _+3.93 25.33 4.1.15
0.00 4. 0.00 0.00__ 0.00 3.30 __0.58 24.83 4. 3.60 24.25 _+1.89 29.40 +_5.98 24.67 +_2.52
0.00 __0.00 0.004-0.00 5.67 _+2.08 20.67 _+2.07 30.75 _+3.86 28.50 _+6.25 19.00 _+3.61
0.00 4. 0.00
0.00 4- 0.00
2.67 4-1.15
22.50 4- 4.50
26.78 _+5.40
22.50 4- 4.40
rostrocaudal extent of the midbrain the superficial layers of the superior colliculus are labeled densely with Fos-LI (Fig. 3F). Labeled cells are also seen in the various regions of the periaqueductal gray, the dorsal tegmental, and subceruleus nuclei (not shown in figures).
Injections of 1.0-1.4 mg / kg of nicotine Injections of 1.0 or 1.4 mg/kg of nicotine resulted in labeling of cells in MT, interpeduncular nucleus and the superior colliculus, but not in SNpc or SNpr, nucleus parabrachialis pigmentosus, rostral linear nucleus, interfascicular nucleus or the nucleus paranigralis. Injections of doses higher than 0.4 mg/kg of nicotine did not alter either the distribution pattern or the number of Fos-positive cells in these nuclei.
Mecamylamine injections Nicotine-induced c-los expression is clearly blocked by 1 mg/kg dose of mecamylamine administered 30 min prior to nicotine injections. The IP and MT are free of any cells labeled with Fos-LI (Fig. 3E), but few cells with Fos-LI are seen in the superficial layer of the superior colliculus, thereby suggesting that expression of Fos in some cell type in the superfical layer of superior colliculus may not be mediated by nicotinic receptors.
Quantitative analysis The results from the present study show that the number of Fos-reactive cells are significantly increased in those rats that received nicotine than in control rats (Table I). A dose of 0.1 m g / k g of nicotine resulted in marginal increase in the number of Fos-reactive cells in several midbrain nuclei, but a dose of 0.4 mg/kg of
nicotine resulted in significant increase in the number of Fos-reactive cells in these midbrain nuclei. Mecamylamine completely abolished Fos-LI in almost all of the midbrain nuclei. A group of rats that were sacrificed 4 h after 0.4 mg/kg of nicotine injections continued to exhibit significant number of Fos-immunoreactive cells in the superior colliculus, MTN and the interpeduncular nucleus. DISCUSSION
General comments The results show that neurons labeled with Fos-LI were noted prominently after 2 h. Significant numbers of neurons with Fos-LI were noted in several VTA nuclei even 4 h after nicotine injections (Table I). This pattern is consistent with the general pattern of expression of Fos protein as reported by several other investigators 10,43.
Labeling pattern in saline and drug-naive and saline-injected animals The present study shows that even in drug-naive and saline-treated 'control' animals significant numbers of neurons with Fos-LI were noted in the supramamillary nucleus, peripeduncular nucleus, an area dorsal to and medial to medial geniculate nucleus, but not in the interpeduncular nucleus, MT or SNpc. This pattern of expression of neurons within these areas in saline-injected animals is similar to that reported by Deutch et al. 8. This level of expression of the c-fos protooncogene may reflect the basal level of excitation of these cells in 'control' animals, or may reflect neuronal activation associated with injection a n d / o r animal handling procedures.
168 Distribution pattern of neurons with Fos-LI in nicotineinjected animals Nicotine dose and c-fos activation London et al. 28, using the 2-deoxyglucose method, noticed that the rate of utilization of glucose increased proportionately in many areas of CNS with gradually increasing doses of nicotine. A very low dose of nicotine (0.1 mg/kg) provided a marginal behavioral response and an associated increase in glucose utilization in few regions of CNS, but a sustained behavioral response and an increased level of utilization of glucose in many other areas of CNS was noticed after a higher dose (0.3 mg/kg). Some areas of the brain showed a greater rate of glucose utilization with a 1.0 m g / k g dose than with a 0.3 m g / k g dose, but the rate of glucose utilization was similar when 1.0 m g / k g or 1.75 m g / k g of s.c. nicotine was injected. The glucose utilization rate in VTA was 45% above the control level with 0.3 m g / k g s.c. of nicotine. In our experiments, the saline-injected 'control' animals did not contain any cells within the interpeduncular nucleus and MT. But a nicotine dose as low as 0.1 m g / k g resulted in labeling of many neurons in the superficial layer of the superior colliculus, the interpeduncular nucleus and MT. There was a significant increase in the number and intensity of staining of neurons with Fos-LI in these VTA nuclei after 0.3 and 0.4 m g / k g doses of nicotine. Doses higher than 0.4 mg/kg, viz., 0.8 mg. 1.0 mg and 1.4 mg kg, did not increase the number or the intensity of staining of the labeled neurons, or alter the distribution pattern of neurons expressing Fos-LI in VTA (Table I). Nicotine, Fos expression and time course of metabolic response to nicotine London et al. 2s, using 2-deoxyglucose methods, showed that extraneurally administered nicotine enters the brain and causes significant behavioral effects quickly. Within 2 min after administration, nicotine binds to several brain regions that are recognized to contain high density nicotine binding sites and causes significant increase in local cerebral glucose utilization (LCGU) in these areas. The LCGU dissipated within 15 min after nicotine administration in almost all brain regions except the superior colliculus, in which the LCGU lasted for 120 min. While the L C G U declined within 15 min, the behavioral effects appeared to last longer after higher doses of nicotine 28. The present study shows that Fos-reactive neurons in the superior colliculus and MT remain even 4 h after nicotine injections. The pattern of persistence of Fos-LI in
neurons of the midbrain several hours after administration of single dose of nicotine appears to parallel the pattern of persistence of Fos immunoreactivity after a single dose of cocaine. Young et al. 43 noticed that after a single dose of 30 m g / k g of cocaine, Fos immunoreactivity persisted even upto 24 h after drug treatment. These studies suggest that a single dose nicotine may produce effects that are long lasting at molecular levels, and it is possible that these long lasting effects on the expression of c-fos oncogene and other genes may contribute to nicotine dependence. Correlation of distribution pattern of Fos-LI and nicotine binding sites in the midbrain A significant increase in the rate of glucose utilization has been observed in the infundibular nucleus, the superior colliculus and several other midbrain nuclei after 0.3 m g / k g of s.c. nicotine 2s. These nuclei are well recognized to contain very dense high affinity [3H] nicotine and [3H]acetylcholine binding sites 6 and also correspond to areas that are densely immunolabeled by monoclonal antibodies (mAb 35.747, mab 290 and mAb 27037) to an a-subunit of nicotinic receptors. A quantitative in vivo [3H]nicotine binding study by London et al. 26 clearly showed that the interpeduncular nucleus had the some of the highest densities of nicotine binding sites (27.8 f m o l / m g tissue) of any brain region assayed and the superficial layer of the superior colliculus was the third highest in density (12.9 f m o l / m g tissue) of nicotine binding sites. Consistent with these observations, acute injections of nicotine resulted in a significant increase in the number of c-fos expressing neurons in the MT, interpeduncular nucleus and the superior colliculus. The pattern of expression of Fos after acute nicotine injections is similar to the pattern of nicotine binding sites in VTA except in SNpc 6. Subcutaneous and iontophoretic injections of nicotine into VTA and SNpc significantly increase the firing rate of the DA neurons z5~29. This increased firing rate of A10 and A9 DA neurons was not altered by i.v. apomorphine but were reversed by administration of mecamylamine, thereby suggesting that the nicotinergic effects are probably not mediated by dopaminergic receptors, but by nicotinergic receptors z~. Neuropharmacological studies have clearly demonstrated that nicotinic receptors are expressed in both the soma and the nerve terminals of mesolimbic and nigrostriatal neurons 5'~4. Wada et al. 4° using in situ hybridization techniques have localized mRNA for ~3-, a4- and /32-subunits of nicotinic receptors in the soma of several nuclei of the ventral tegmental areas, including VTA, SNpc, MT and the interpeduncular nucleus.
169 In spite of these lines of evidence which suggest that nicotine causes excitation of VTA and SNpc neurons, much to our surprise, even a very high but subconvulsive dose of nicotine (1.4 mg/kg, s.c.), failed to induce Fos-LI in SNpc and most of the subdivisions of VTA. The possibility that nicotine binds to SNpc and other DA cells of VTA and induces expression of some other member of the immediate early genes family but not c-fos cannot be excluded.
Among the VTA subnuclei, nicotine induced Fos-LI only in CLI Even though most of the VTA subnuclei did not contain Fos-LI, the caudal linear subnucleus of VTA (CLI) demonstrated several cells with Fos-LI. CLI has been shown to contain DA neurons 34, and DA neurons colocalized with cholecystokinin (CCK) and neurotensin (NT). The number of neurons as well as the intensity of expression of mRNA of NT is higher in CLI than any other VTA subnucleus 23. It is of interest to note that NT is localized prominently in various autonomic regions of the CNS 38 and causes significant changes in the heartrate and cardiovascular response when injected intracerebroventricularly. Stress and stress-inducing chemicals actively induce c-fos expression intensely in CLI 8. Acute injections of nicotine lead to significant autonomic responses zS. The possibility that some of the autonomic responses of nicotine may be mediated by CLI and other subnuclei that contain neurotensin remains to be explored.
Nicotine and accessory optic system A major observation of this study is the presence of Fos-reactive neurons in several nuclei of the midbrain that are related to the accessory optic system (AOS). The AOS has been studied in great detail in several vertebrates 3'12'13'35. The AOS plays a major role in stabilizing the drift of an image on the retina 13'35. The different subdivisions of the AOS receive direction selective input from the retina 13 and contain a high density of [3H]nicotine binding sites 6'26'27. Nicotine injections result in a dramatic increase in LCGU in AOS and enucleation abolishes nicotine-induced glucose utilization in these subnuclei 27. In our study, acute injections of nicotine induce expression of Fos very prominently in the superior colliculus and the medial terminal nucleus. The lateral and dorsal terminal nuclei also contained several neurons with Fos-LI, whereas cells with Fos-LI were absent in the ventral tegmental relay zone. The precise role played by the intense activation of the AOS by nicotine remains to be elucidated.
CONCLUSIONS The current study suggests several important aspects of nicotinergic effects on the midbrain neurons: (i) Nicotine induces Fos expression in neurons of several midbrain nuclei that are activated by stressful stimuli (namely supramamiUary nucleus and CLI). (ii) A nicotine dose as low as 0.1 mg/kg s.c., induces Fos expression in many regions of the midbrain, but 0.4 mg/kg provides the maximum response. Doses of nicotine higher than 0.4 mg do not alter the pattern or increase the number of neurons expressing Fos in the midbrain. (iii) Acute injections of nicotine stimulate neurons of several brain stem nuclei that respond to visual and visuomotor stimuli. (iv) Acute injections of nicotine induce c-fos activation mostly in non-dopaminergic neurons of the midbrain. Acknowledgements. Supported by The Smokeless Tobacco Research Council, New York. H.K. is a Visiting Scientist from the Nihon University School of Dentistry at Matsudo, Matsudo, Chiba, Japan.
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Molecular Brain Research, 20 (1993) 162-170 ~ 1993 Elsevier Science Publishers B.V. All rights reserved 0169-328x/93/$06.00
BRESM 70654
Acute nicotine injections induce c-fos mostly in non-dopaminergic neurons of the midbrain of the rat Ying Pang, Hideo Kiba and A. Jayaraman Department of Neurology. LSU School of Medicine, New Orleans, LA 70112 (USA) (Accepted 20 April 1993)
Key words: Protooncogene; Reinforcement: Mecamylamine; Fos; Accessory optic system: Ventral tegmental area: Dopamine; Nicotine
Induction of c-los gene is an immediate and early response in the cascade of molecular events that ultimately lead to long-term alterations in gene expression in neurons. The psychomotor stimulant and positive reinforcing effects of nicotine have been speculated to be mediated by the dopaminergic neurons of the ventral tegmental area (VTA). To identify the precise subsets of VTA neurons of the rat that mediate the acute nicotinergic effects, the pattern of expression of c-los gene was mapped using immunocytochemical methods. Acute nicotine injections resulted in prominent Fos-like immunoreactivity (-LI) in the medial terminal nucleus of the accessory optic system, the interpeduncular nucleus, and in the caudal linear subnucleus of VTA. The neurons of other VTA subnuclei, viz., the rostral linear, paranigralis, nucleus parabrachialis pigmentosus, and nucleus interfascicularis or the substantia nigra pars compacta did not contain any cells with Fos-LI. Mecamylamine abolished Fos-LI in most of the VTA neurons. These results suggest that acute nicotine injections induce c-los expression mostly in non-dopaminergic neurons of the ventral tegmental area of the rat and that nicotine induces c-los most intensely in the interpeduncular nucleus, the superior colliculus, and several other subnuclei of the accessory optic system.
INTRODUCTION
VTA DA soma and the terminals in the nucleus accumbens4,5,6,14,18,22.2529,4o.
Nicotine is a psychomotor stimulant and a positive reinforcer ~,15.20. The precise CNS sites and the mechanisms with which nicotine enhances the positive effects and leads to repeated self-administrative behavior are poorly understood. Compelling evidence from several laboratories has lead to the establishment of a working hypothesis that the mesolimbic dopaminergic (DA) neurons of the ventral tegmental area (VTA) and their projections play a major role in mechanisms underlying emotion, motivation, reward and reinforcement behavior 1t'24'42. Several studies indeed suggest that nicotine has an excitatory effect on the midbrain dopaminergic neurons, nicotine stimulates the A10 dopaminergic neurons of the VTA more potently and selectively than the A9 cells in the substantia nigra pars compacta (SNpc), and these stimulatory effects appear to be mediated by the nicotinic receptors localized on the
The mesencephalic DA neurons may be divided into many subdivisions and these individual subnuclei have afferent and efferent projections 12'1'~'3°'33'36 and neurochemical organization that is specific to each of the subnuclei 23"34'4t. It is possible that each of the subnuclei may contribute to different aspects of the 'reward" experience. As a crucial step towards understanding the neurochemical and molecular mechanisms underlying the interactions between nicotine and the midbrain DA neurons, it is important to identify the specific subnuclei of the ventral tegmental neurons that mediate the nicotinergic effects. In order to achieve this goal, experiments were conducted to define the distribution of the VTA cells expressing the protooncogene c-los after acute injections of nicotine. The c-los gene encodes a nuclear protein transcription modulator (Fos) that exhibits DNA binding in
Correspondence." A. Jayaraman, Department of Neurology, LSU School of Medicine, 1542 Tulane Ave., New Orleans, LA 70112, USA. Fax: (1) (504) 568-4084.
163 vitro. T h e c-los g e n e is o n e o f several i m m e d i a t e e a r l y g e n e s d i s t i n g u i s h e d by t h e i r r a p i d a n d t r a n s i e n t t r a n s c r i p t i o n a l activation. T h e g e n e c-fos is i n d u c e d by a
variety of e x t r a c e l l u l a r stimuli. T h e s e i n c l u d e electrically a n d n e u r o c h e m i c a l l y i n d u c e d seizures, electrical, physiological, n o c i c e p t i v e a n d m e t a b o l i c stimulation. In
B
A
,TN
C
D
~TN
Fig. 1. Fos-LI was noted in neurons of the supramamillary nucleus (SUM), retrorubral (RRF) and peripeduncular areas, lateral and dorsal terminal nuclei even in saline and drug-naive rats (A,B) as well as in the saline-injected 'control' rats (C,D). Note the absence of Fos-LI in the superior colliculus and the SN complex.
164 Several studies have shown that induction of c-fos can be used as a marker, with single-cell specificity, to observe the temporal and spatial involvement of cer-
PC12 cells, c-los is induced by membrane depolarization and stimulation of nicotinic acetylcholine receptors (reviewed in ref. 9).
A
8
TN
D
C;
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* ...;..
•¢.,.~.. D'rN
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-.o ~,~o
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Fig. 2. After an injection of 0.4 mg of s.c. nicotine, neurons of the medial terminal nucleus of the accessory optic system (MT) (A) and the interpeduncular nucleus (IP) (B,C,D) contained cells labeled intensely with Fos-LI. Note that some cells in the lateral terminal nucleus (A) and some in the dorsal terminal nucleus (C) are labeled with Fos-LI. A m o n g the many subdivisions of V T A the most prominent labeling was noted in caudal liner nucleus (CLI) (C,D), but Fos-LI was not seen in the interfascicular, paranigralis nuclei and nucleus parabrachialis pigmentosus of V T A (A,B). The superior collieulus and the periaqueductal gray were densely labeled with Fos-LI ( A - D ) in all experiments.
165 tain subsets of neurons following seizures. Using similar principles, cocaine- and amphetamine-responsive cells have been mapped in the striatum ~7. MATERIALS AND METHODS Experiments were conducted in several groups of male SpragueDawley rats (250-350 g). One group of control rats (n = 3) was saline
and drug naive whereas the second group of control rats (n = 4) was nicotine naive but received saline 1.0 ml alone. The experimental group of rats received subcutaneous injections of 0.1 mg (n = 3), 0.4 mg (n = 5), 0.8 mg (n = 4), 1.0 mg (n = 5) and 1.4 mg/kg (n = 3) of nicotine (Sigma, St. Louis, MO) in 0.9% NaCI (pH 7.0+0.2). The selection of different doses of nicotine adopted in our study was based on nicotine doses used by London et al. 26'27'28 in their studies of nicotine-induced local cerebral glucose utilization patterns. Another group (n = 3) received 1 mg/kg of mecamylamine (Sigma, St. Louis, MO) 30 min prior to 0.4 mg/kg of nicotine injections. The
Fig. 3. Photomicrographs show promment Fos-immunoreactive neurons in MT (A), interpeduncular nucleus (C) caudal linear subnucleus of VTA (D) and the superior colliculus (F). Note that the VTA subnuclei and substantia nigra do not contain any neurons with Fos-LI. Experiments using in situ hybridization methods with an oligonucleotide probe directed against rat cDNA for TH clearly show prominent hybridization signal in the VTA and substantia nigra pars compacta (SNC) (B), but not in MT. Nicotine-induced c-fos expression in VTA was blocked by mecamylamine (E) administered 30 min prior to nicotine injections.
166 mecamylamine dose was selected on the basis of the the dose of the drug adopted in the studies of Rosecrans et al. 32. The majority of the animals were sacrificed 2 h after nicotine injections, but one group of rats (n = 3) received 0.4 mg/kg of nicotine and were sacrificed 4 h after nicotine injections. The control and treated rats were anesthetized with sodium pentobarbital (25 mg/kg., i.p.,) and were perfused intracardially with a solution containing 4% paraformaldehyde in sodium phosphate pH 7.4. The brains were removed and postfixed in perfusate for about 1 h. The brains were blocked and stored overnight in sucrose solution. Subsequently the brains were cut into 40/.~ thick sections. The sections were then processed for the standard avidin-biotin immunohistochemical procedure 19 using peroxidase Vectastain Elite ABC Kit (Vector laboratories, Burlingame, CA) and by following the protocol suggested by the manufacturer. For detecting Fos the sections were incubated in 1:500 to 1:1000 concentration of a polyclonal antibody for Fos (PC05, Oncogene Science, Uniondale, NY). Control sections were incubated in the presence of Fos peptides (PC09, PC10, Oncogene Science, Uniondale, NY) to which the antibodies were raised. In few cases, adjacent sections were processed for Fos-like immunoreactivity (Fos-LI) and for localizing neurons that contain mRNA for rat tyrosine hydroxylase with in situ hybridization methods. The oligoprobes used in these experiments were directed against the nucleotide sequence of 1441-1485 of the rat tyrosine hydroxylase cDNA2. The sections were studied with darkfield and brightfield microscopy, and projection drawings were made using camera lucida. The cytoarchitectonic details of various nuclei of the midbrain and especially VTA were defined in adjacent sections counterstained with cresyl violet. The atlas of Paxinos and Watson 31 was used to identify various mesencephalic nuclei. For quantitative analyses, the number of Fos positive cells was ascertained by manually counting neurons with Fos-Ll within a 10 x 10 ~2 area of the superfical layers of the superior colliculus, MT and the interpeduncular nucleus under 40 x magnification. RESULTS
Distribution pattern of cells with Fos-LI in saline and drug naiL,e rats F o s - L I is n o t e d in m a n y n e u r o n s of t h e m i d b r a i n even in saline a n d d r u g naive rats. M a j o r i t y of t h e s e n e u r o n s are seen in t h e s u p r a m a m i l l a r y nucleus. F e w n e u r o n s with F o s - L I are also n o t e d in t h e p e r i p e d u n c u lar region, b u t n o n e are seen in t h e i n t e r p e d u n c u l a r nucleus, s u p e r i o r colliculus, V T A o r the s u b s t a n t i a nigra c o m p l e x (Fig. 1A, B).
Distribution pattern of cells with Fos-LI in saline-treated rats F o s - L I is n o t e d in m a n y n e u r o n s of t h e m i d b r a i n in saline t r e a t e d animals. Cells of the s u p r a m a m i l l a r y nucleus are p r o m i n e n t l y l a b l e d with F o s - L I . N e u r o n s l a b e l e d with F o s - L I a r e also n o t i c e d in an a r e a l a t e r a l to the s u p r a m a m i l l a r y nucleus and e x t e n d i n g in an a r e a ventral to m e d i a l lemniscus a n d v e n t r o m e d i a l to m e d i a l g e n i c u l a t e nucleus in t h e p e r i p e d u n c u l a r region. A m o n g the various subnuclei o f the V T A , few cells l a b e l e d with F o s - L I are n o t e d in the rostral l i n e a r
nucleus, i n t e r f a s i c u l a r nucleus a n d c a u d a l l i n e a r nucleus, but cells with F o s - L I are a b s e n t in the nucleus parabrachialis pigmentosus, paranigratis, interpeduncular nucleus, superficial layers o f the s u p e r i o r colliculus, a n d m e d i a l t e r m i n a l nucleus of the optic tract (Fig.
1C,D). Distribution pattern of Fos-LI in nicotine-injected rats Injections of O.1 mg / kg of nicotine A f t e r 0.1 m g / k g o f s.c., m a n y cells in the s u p r a m a m i l l a r y nucleus a n d the p e r i p e d u n c u l a r regions are l a b e l e d with F o s - L I . In a d d i t i o n significant n u m b e r o f F o s - r e a c t i v e cells a r e also n o t e d in the superficial layers o f the s u p e r i o r colliculus, the m e d i a l t e r m i n a l nucleus o f the accessory optic system (MT), a n d the i n t e r p e d u n c u l a r nucleus (not shown in figures).
Injections of 0.4 mg / kg of nicotine T h e p a t t e r n of d i s t r i b u t i o n o f n e u r o n s with F o s - L I in V T A b e c o m e m o r e clearly d e f i n e d as the n u m b e r of F o s - r e a c t i v e cells i n c r e a s e d d r a m a t i c a l l y w h e n t h e dose of nicotine injected is i n c r e a s e d to 0.4 m g / k g s.c. A f t e r 0.4 m g / k g o f s.c nicotine, at the level of t h e p o s t e r i o r h y p o t h a l a m u s , t h e s u p r a m a m i l l a r y nucleus c o n t a i n e d significant n u m b e r o f p r o m i n e n t l y l a b e l e d cells with F o s - L I . A t the rostral most level o f V T A , the m o s t i n t e n s e l a b e l i n g is n o t e d in M T (Figs. 2 A a n d 3A). T h e M T e x t e n d s f r o m an a r e a m e d i a l to t h e m e d i a l - m o s t a r e a of s u b s t a n t i a nigra p a r s c o m p a c t a ( S N p c ) a n d spans d o r s o l a t e r a l l y into a region v e n t r a l to m e d i a l l e m n i s c u s a n d p a r a l l e l to the d o p a m i n e r g i c n e u r o n s of SNpc. O n t h e basis of n e u r a l connectivity p a t t e r n M T has b e e n i d e n t i f i e d to have v e n t r a l a n d d o r s a l subdivisions. L a b e l e d cells a r e seen t h r o u g h o u t the r o s t r o c a u dal e x t e n t of b o t h ventral a n d d o r s a l subdivisions of M T (Figs. 2 A a n d 3A). A t rostral V T A levels, cells l a b e l e d with F o s - L I are few in the nucleus interfascicularis, a n d p a r a n i g r a l i s , but n o n e a r e n o t e d in n e u r o n s o f nucleus p a r a b r a c h i a l i s p i g m e n t o s u s , a n d s u b s t a n t i a nigra p a r s c o m p a c t a ( S N p c ) which c o n t a i n m R N A for tyrosine hydroxylase (Figs. 2A, B a n d 3A,B). A t m i d a n d c a u d a l V T A levels, cells with F o s - L I are l a b e l e d intensely in the i n t e r p e d u n c u l a r nucleus. T h e labeling p a t t e r n is n o t e d t h r o u g h o u t t h e r o s t r o c a u d a l extent o f this nucleus, b u t t h e rostral a n d apical divisions of t h e nucleus c o n t a i n e d m o r e cells l a b e l e d with F o s - L I t h a n the i n t e r m e d i a t e a n d l a t e r a l divisions (Figs. 2B,D a n d 3C). A m o n g the m a n y subdivisions o f V T A the most p r o m i n e n t labeling is n o t e d in c a u d a l l i n e a r nucleus ( C L I ) especially at the m i d a n d c a u d a l levels of the nucleus (Figs. 2C,D and 3D). T h r o u g h o u t the entire
167 TABLE I
Two hours post injections Saline and nicotine-naive (n = 3) Saline (n = 4) 0.1 m g / k g nicotine s.c. (n = 3) 0.4 m g / k g nicotine s.c. (n = 5) 0.8 m g / k g nicotine s.c. (n = 4) 1.0 m g / k g nicotine s.c. (n = 5) 1.4 m g / k g nicotine s.c. (n = 3) Mecamylamine + nicotine (1.0 r n g / k g + 0.4 m g / k g ) (n = 3) Four hours post injections 0.4 m g / k g nicotine s.c. (n = 3)
MT (mean 4- S.D.)
IP (mean 4- S.D.)
SC (mean 4- S.D.)
0.00 +_0.00 0.00 :t: 0.00 5.00 _+1.00 23.67 4. 4.32 24.50 _+2.08 25.33 _+3.93 25.33 4.1.15
0.00 4. 0.00 0.00__ 0.00 3.30 __0.58 24.83 4. 3.60 24.25 _+1.89 29.40 +_5.98 24.67 +_2.52
0.00 __0.00 0.004-0.00 5.67 _+2.08 20.67 _+2.07 30.75 _+3.86 28.50 _+6.25 19.00 _+3.61
0.00 4. 0.00
0.00 4- 0.00
2.67 4-1.15
22.50 4- 4.50
26.78 _+5.40
22.50 4- 4.40
rostrocaudal extent of the midbrain the superficial layers of the superior colliculus are labeled densely with Fos-LI (Fig. 3F). Labeled cells are also seen in the various regions of the periaqueductal gray, the dorsal tegmental, and subceruleus nuclei (not shown in figures).
Injections of 1.0-1.4 mg / kg of nicotine Injections of 1.0 or 1.4 mg/kg of nicotine resulted in labeling of cells in MT, interpeduncular nucleus and the superior colliculus, but not in SNpc or SNpr, nucleus parabrachialis pigmentosus, rostral linear nucleus, interfascicular nucleus or the nucleus paranigralis. Injections of doses higher than 0.4 mg/kg of nicotine did not alter either the distribution pattern or the number of Fos-positive cells in these nuclei.
Mecamylamine injections Nicotine-induced c-los expression is clearly blocked by 1 mg/kg dose of mecamylamine administered 30 min prior to nicotine injections. The IP and MT are free of any cells labeled with Fos-LI (Fig. 3E), but few cells with Fos-LI are seen in the superficial layer of the superior colliculus, thereby suggesting that expression of Fos in some cell type in the superfical layer of superior colliculus may not be mediated by nicotinic receptors.
Quantitative analysis The results from the present study show that the number of Fos-reactive cells are significantly increased in those rats that received nicotine than in control rats (Table I). A dose of 0.1 m g / k g of nicotine resulted in marginal increase in the number of Fos-reactive cells in several midbrain nuclei, but a dose of 0.4 mg/kg of
nicotine resulted in significant increase in the number of Fos-reactive cells in these midbrain nuclei. Mecamylamine completely abolished Fos-LI in almost all of the midbrain nuclei. A group of rats that were sacrificed 4 h after 0.4 mg/kg of nicotine injections continued to exhibit significant number of Fos-immunoreactive cells in the superior colliculus, MTN and the interpeduncular nucleus. DISCUSSION
General comments The results show that neurons labeled with Fos-LI were noted prominently after 2 h. Significant numbers of neurons with Fos-LI were noted in several VTA nuclei even 4 h after nicotine injections (Table I). This pattern is consistent with the general pattern of expression of Fos protein as reported by several other investigators 10,43.
Labeling pattern in saline and drug-naive and saline-injected animals The present study shows that even in drug-naive and saline-treated 'control' animals significant numbers of neurons with Fos-LI were noted in the supramamillary nucleus, peripeduncular nucleus, an area dorsal to and medial to medial geniculate nucleus, but not in the interpeduncular nucleus, MT or SNpc. This pattern of expression of neurons within these areas in saline-injected animals is similar to that reported by Deutch et al. 8. This level of expression of the c-fos protooncogene may reflect the basal level of excitation of these cells in 'control' animals, or may reflect neuronal activation associated with injection a n d / o r animal handling procedures.
168 Distribution pattern of neurons with Fos-LI in nicotineinjected animals Nicotine dose and c-fos activation London et al. 28, using the 2-deoxyglucose method, noticed that the rate of utilization of glucose increased proportionately in many areas of CNS with gradually increasing doses of nicotine. A very low dose of nicotine (0.1 mg/kg) provided a marginal behavioral response and an associated increase in glucose utilization in few regions of CNS, but a sustained behavioral response and an increased level of utilization of glucose in many other areas of CNS was noticed after a higher dose (0.3 mg/kg). Some areas of the brain showed a greater rate of glucose utilization with a 1.0 m g / k g dose than with a 0.3 m g / k g dose, but the rate of glucose utilization was similar when 1.0 m g / k g or 1.75 m g / k g of s.c. nicotine was injected. The glucose utilization rate in VTA was 45% above the control level with 0.3 m g / k g s.c. of nicotine. In our experiments, the saline-injected 'control' animals did not contain any cells within the interpeduncular nucleus and MT. But a nicotine dose as low as 0.1 m g / k g resulted in labeling of many neurons in the superficial layer of the superior colliculus, the interpeduncular nucleus and MT. There was a significant increase in the number and intensity of staining of neurons with Fos-LI in these VTA nuclei after 0.3 and 0.4 m g / k g doses of nicotine. Doses higher than 0.4 mg/kg, viz., 0.8 mg. 1.0 mg and 1.4 mg kg, did not increase the number or the intensity of staining of the labeled neurons, or alter the distribution pattern of neurons expressing Fos-LI in VTA (Table I). Nicotine, Fos expression and time course of metabolic response to nicotine London et al. 2s, using 2-deoxyglucose methods, showed that extraneurally administered nicotine enters the brain and causes significant behavioral effects quickly. Within 2 min after administration, nicotine binds to several brain regions that are recognized to contain high density nicotine binding sites and causes significant increase in local cerebral glucose utilization (LCGU) in these areas. The LCGU dissipated within 15 min after nicotine administration in almost all brain regions except the superior colliculus, in which the LCGU lasted for 120 min. While the L C G U declined within 15 min, the behavioral effects appeared to last longer after higher doses of nicotine 28. The present study shows that Fos-reactive neurons in the superior colliculus and MT remain even 4 h after nicotine injections. The pattern of persistence of Fos-LI in
neurons of the midbrain several hours after administration of single dose of nicotine appears to parallel the pattern of persistence of Fos immunoreactivity after a single dose of cocaine. Young et al. 43 noticed that after a single dose of 30 m g / k g of cocaine, Fos immunoreactivity persisted even upto 24 h after drug treatment. These studies suggest that a single dose nicotine may produce effects that are long lasting at molecular levels, and it is possible that these long lasting effects on the expression of c-fos oncogene and other genes may contribute to nicotine dependence. Correlation of distribution pattern of Fos-LI and nicotine binding sites in the midbrain A significant increase in the rate of glucose utilization has been observed in the infundibular nucleus, the superior colliculus and several other midbrain nuclei after 0.3 m g / k g of s.c. nicotine 2s. These nuclei are well recognized to contain very dense high affinity [3H] nicotine and [3H]acetylcholine binding sites 6 and also correspond to areas that are densely immunolabeled by monoclonal antibodies (mAb 35.747, mab 290 and mAb 27037) to an a-subunit of nicotinic receptors. A quantitative in vivo [3H]nicotine binding study by London et al. 26 clearly showed that the interpeduncular nucleus had the some of the highest densities of nicotine binding sites (27.8 f m o l / m g tissue) of any brain region assayed and the superficial layer of the superior colliculus was the third highest in density (12.9 f m o l / m g tissue) of nicotine binding sites. Consistent with these observations, acute injections of nicotine resulted in a significant increase in the number of c-fos expressing neurons in the MT, interpeduncular nucleus and the superior colliculus. The pattern of expression of Fos after acute nicotine injections is similar to the pattern of nicotine binding sites in VTA except in SNpc 6. Subcutaneous and iontophoretic injections of nicotine into VTA and SNpc significantly increase the firing rate of the DA neurons z5~29. This increased firing rate of A10 and A9 DA neurons was not altered by i.v. apomorphine but were reversed by administration of mecamylamine, thereby suggesting that the nicotinergic effects are probably not mediated by dopaminergic receptors, but by nicotinergic receptors z~. Neuropharmacological studies have clearly demonstrated that nicotinic receptors are expressed in both the soma and the nerve terminals of mesolimbic and nigrostriatal neurons 5'~4. Wada et al. 4° using in situ hybridization techniques have localized mRNA for ~3-, a4- and /32-subunits of nicotinic receptors in the soma of several nuclei of the ventral tegmental areas, including VTA, SNpc, MT and the interpeduncular nucleus.
169 In spite of these lines of evidence which suggest that nicotine causes excitation of VTA and SNpc neurons, much to our surprise, even a very high but subconvulsive dose of nicotine (1.4 mg/kg, s.c.), failed to induce Fos-LI in SNpc and most of the subdivisions of VTA. The possibility that nicotine binds to SNpc and other DA cells of VTA and induces expression of some other member of the immediate early genes family but not c-fos cannot be excluded.
Among the VTA subnuclei, nicotine induced Fos-LI only in CLI Even though most of the VTA subnuclei did not contain Fos-LI, the caudal linear subnucleus of VTA (CLI) demonstrated several cells with Fos-LI. CLI has been shown to contain DA neurons 34, and DA neurons colocalized with cholecystokinin (CCK) and neurotensin (NT). The number of neurons as well as the intensity of expression of mRNA of NT is higher in CLI than any other VTA subnucleus 23. It is of interest to note that NT is localized prominently in various autonomic regions of the CNS 38 and causes significant changes in the heartrate and cardiovascular response when injected intracerebroventricularly. Stress and stress-inducing chemicals actively induce c-fos expression intensely in CLI 8. Acute injections of nicotine lead to significant autonomic responses zS. The possibility that some of the autonomic responses of nicotine may be mediated by CLI and other subnuclei that contain neurotensin remains to be explored.
Nicotine and accessory optic system A major observation of this study is the presence of Fos-reactive neurons in several nuclei of the midbrain that are related to the accessory optic system (AOS). The AOS has been studied in great detail in several vertebrates 3'12'13'35. The AOS plays a major role in stabilizing the drift of an image on the retina 13'35. The different subdivisions of the AOS receive direction selective input from the retina 13 and contain a high density of [3H]nicotine binding sites 6'26'27. Nicotine injections result in a dramatic increase in LCGU in AOS and enucleation abolishes nicotine-induced glucose utilization in these subnuclei 27. In our study, acute injections of nicotine induce expression of Fos very prominently in the superior colliculus and the medial terminal nucleus. The lateral and dorsal terminal nuclei also contained several neurons with Fos-LI, whereas cells with Fos-LI were absent in the ventral tegmental relay zone. The precise role played by the intense activation of the AOS by nicotine remains to be elucidated.
CONCLUSIONS The current study suggests several important aspects of nicotinergic effects on the midbrain neurons: (i) Nicotine induces Fos expression in neurons of several midbrain nuclei that are activated by stressful stimuli (namely supramamiUary nucleus and CLI). (ii) A nicotine dose as low as 0.1 mg/kg s.c., induces Fos expression in many regions of the midbrain, but 0.4 mg/kg provides the maximum response. Doses of nicotine higher than 0.4 mg do not alter the pattern or increase the number of neurons expressing Fos in the midbrain. (iii) Acute injections of nicotine stimulate neurons of several brain stem nuclei that respond to visual and visuomotor stimuli. (iv) Acute injections of nicotine induce c-fos activation mostly in non-dopaminergic neurons of the midbrain. Acknowledgements. Supported by The Smokeless Tobacco Research Council, New York. H.K. is a Visiting Scientist from the Nihon University School of Dentistry at Matsudo, Matsudo, Chiba, Japan.
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