Striato-nigral dynorphin and substance P pathways in ... - Springer Link

Ex mental BranResearch

Exp Brain Res (1986) 64:169-192

9 Springer-Verlag 1986

Striato-nigral dynorphin and substance P pathways in the rat I. Biochemical and immunohistochemical studies I. Christensson-Nylander1, M. Herrera-Marschitz2, W. Staines3, T. H6kfelt3, L. Terenius 1, U. Ungerstedt 2, C. Cuello4, W.H. Oertel 5, an d M. Goldstein6 1 Departmentof Pharmacology,Universityof Uppsala, S-75124 Uppsala, Sweden 2 Departmentsof Pharmacologyand 3 Histology,KarolinskaInstitutet, S-10401 Stockholm,Sweden 4 Departmentof Pharmacologyand Therapeutics,McGillUniversity,Montreal, Canada 5 Departmentof Neurology,TechnicalUniversityof Miinchen,D-8000 Miinchen,Federal Republicof Germany 6 Departmentof Psychiatry,New York University,MedicalCenter, New York, NY, USA

Summary. The effect of striatal ibotenic acid lesions on dynorphin-, substance P- and enkephalin-like immunoreactivities in the substantia nigra has been studied with immunohistochemistry as well as biochemistry. A comparison was made with the effects produced by intranigral ibotenic acid lesion and by 6-hydroxy-dopamine injection into the medial forebrain bundle. In addition, the effect of the striatal lesions on nigral glutamic acid decarboxylase (GAD)-positive structures was analysed with immunohistochemistry. The effect of the lesions was analysed functionally in the Ungerstedt rotational model, in order to obtain a preliminary evaluation of the extent of the lesions. The striatal lesions produced a parallel depletion of dynorphin and substance P levels in the substantia nigra, pars reticulata, ipsilateral to the treated side, which was dependent upon the extent and location of the lesion. Ibotenic acid lesions into the tail and the corpus of the striatum produced stronger nigral-peptide depletion than lesions in the head and the corpus of the striatum. Comparison of placement of lesions and localization of depleted area in the substantia nigra revealed a topographical relationship. Furthermore, the nigral depletion patterns of dynorphin and substance P were similar. The immunohistochemical analysis revealed that also GAD-positive fibers in the pars reticulata to a large extent disappeared after striatal lesions, in parallel to the dynorphin- and substance P-positive fibers. However, the depletion was less pronounced for GAD than for the peptides, probably related to presence of local GABA neurons in the zona reticulata of the substantia nigra. These results indicate that with the types of lesion used in this study it is not possible to provide evidence for a differential localization within the striatum of dynor-

Offprint requests to: I. Christensson-Nylander(addresssee above)

phin-, substance P- and GABA-positive cell bodies projecting to the substantia nigra. The radioimmunoassay showed that (Leu)- but not (Met)enkephalin was affected to the same extent as the dynorphin peptides, supporting the view that (Leu)enkephalin in the pars reticulata of the substantia nigra is derived from proenkephalin B and not from proenkephalin A. In the immunohistochemical analysis (Met)-enkephalin-like immunoreactivity could only be detected in the pars compacta of the substantia nigra and did not seem to be affected by any of the lesions. The striatal lesions produced a behavioural asymmetry, which could be disclosed by stimulating the rats with apomorphine, which produced ipsilateral rotation. The total number and intensity of the rotation were closely correlated to the extent and location of the striatal lesion as well as to the amount of dynorphin and substance P depletion found in the substantia nigra of the treated side. The results provide further evidence for the presence of a dynorphin-containing system with fibers originating mainly in the corpus and tail of the striatum and terminating in the zona reticulata of the substantia nigra and may, similarly to the previously characterized substance P and GABA containing pathways, have a role in the control of motor behaviour.

Key words: Basal ganglia - Transmitters - Neuropeptides - GABA - Enkephalin - Striato-nigral pathways

Introduction The various components of the basal ganglia are interconnected by a number of anatomically characterized neuronal pathways, and many different neurotransmitters have been described in this sys-

170

tern; they include members of each of the three groups of messengers, amines, amino acids and peptides (see reviews by Graybiel and Ragsdale 1983, McGeer et al. 1984). The anatomy and transmitter biochemistry of the substantia nigra has been of particular interest as it is one of the points of outflow of activity from the basal ganglia, via the projections of the pars reticulata (Gale and Casu 1981; Gerfen et al. 1982; Graybiel and Ragsdale 1983; McGeer et al. 1984), and also gives rise, via the projections of the pars compacta, to an ascending dopaminergic projection of well characterized functional and clinical importance (Dahlstr6m and Fuxe 1964; Anden et al. 1966; Ungerstedt 1971a, DiCarlo et al. 1973; Lindvall and Bj6rklund 1974; Poitras and Parent 1978). The striatum constitutes the main source of afferents to the substantia nigra. It has been shown that striatonigral neurons form monosynaptic inputs to nigrostriatal neurons (Somogyi et al. 1979, 1981), which may represent a feed-back loop regulating the firing of nigro-striatal dopamine (DA) neurons, but may also govern efferent activity from the pars reticulata of the substantia nigra (see Graybiel and Ragsdale 1983). The striato-nigral pathway has been shown to be neurochemically heterogenous, containing y-aminobutyric acid (GABA) (Kim et al. 1971; Hattori et al. 1973; Fonnum et al. 1974, 1978; Brownstein et al. 1977; Gale et al. 1977; Jessell et al. 1978; Nagy et al. 1978; Ribak et al. 1979, 1980; Gale and Casu 1981), substance P (SP) (Brownstein et al. 1977; Gale et al. 1977; Hong et al. 1977; Kanazawa et al. 1977; Jessell et al. 1978; Ljungdahl et al. 1978a, b; Staines et al. 1980; Oertel et al. 1981b; McGeer et al. 1984) and, more recently, the opioid peptides derived from the proenkephalin B (prodynorphin) precursor (dynorphin A = DYN A, dynorphin B = DYN B, c~-neoendorphin, [3-neoendorphin) (Vincent et al. 1982a; Palkovits et al. 1984; Zamir et al. 1984a; Fallon et al. 1985). The peptides DYN A, DYN B, ~-neoendorphin and ~-neoendorphin (Goldstein et al. 1979, 1981; Tachibana et al. 1982) are present in high concentrations in the substantia nigra of several species, as compared to other brain structures (Gramsch et al. 1982; Maysinger et al. 1982; Zamir et al. 1983; Pittius et al. 1984; Zamir et al. 1984b-e; ChristenssonNylander and Terenius 1985; Dores et al. 1985). Also the low molecular weight members of the DYN family, DYN A (1-8) and (leucine) enkephalin ((Leu) Enk), have been shown to exist in this structure (Zamir et al. 1984a-b). The opioid pentapeptide (Leu) Enk sequence is present in two different opioid precursors: proenkephalin A (con-

taining one sequence) (Comb et al. 1982; Noda et al. 1982) and proenkephalin B (containing three sequences) (Kakidani et al. 1982). Until recently, only proenkephalin A was thought to act as a true precursor of (Leu) Enk, due to the observation that, in most brain areas, (methionine) enkephalin ((Met) Enk) and (Leu) Enk are present in a ratio of 4 : 1, similar to that found in proenkephalin A. However, in the substantia nigra, the ratio of (Met) Enk to (Leu) Enk is different from that in other regions, with (Leu) Enk as the predominant peptide (ratio 1 : 2), suggesting that it may derive from a different precursor. More evidence that this peptide derives from proenkephalin B rather than proenkephalin A has also recently been presented based on knife lesions of the striato-nigral pathways (Zamir et al. 1984a). With immunohistochemical techniques, DYN immunoreactive cell bodies have been demonstrated in the striatum and a corresponding dense plexus of DYN positive nerve terminals has been described in the substantia nigra, mainly in the zona reticulata (Khachaturian et al. 1982; Vincent et al. 1982b; Weber et al. 1982; Weber and Barchas 1983; Fallon et al. 1985). Knife cuts of the capsula interna, producing lesions of the pathways between striatum and substantia nigra, reduce nigral DYN immunoreactivity, indicating a DYN striato-nigral pathway (Palkovits et al. 1984; Zamir et al. 1984a). Preliminary studies, using ibotenic acid (IBA) injections to destroy striatal cell bodies, have also been shown to reduce DYN immunofluorescence in the substantia nigra (Vincent et al. 1982a). A functional role for DYN in these structures has been suggested by the observation that injections of DYN peptides into the substantia nigra of rats cause a dose dependent contralateral rotational behaviour which can be potentiated by amphetamine (Herrera-Marschitz et al. 1983; 1984; H6kfelt et al. 1984b; Morelli and Di Chiara 1985). The present paper describes the use of chemical lesions to cause defined destruction of striato-nigral or nigro-striatal pathways. The effect of these lesions on DYN A, DYN B, SP, (Leu) Enk and (Met) Enk levels in substantia nigra has been studied immunohistochemically and biochemically. In addition the GABA synthesizing enzyme glutamic acid decarboxylase (GAD) has been investigated with immunohistochemistry. The effects of the lesions were also analysed behaviourally, in order to obtain a direct functional correlate to the immunohistochemical and biochemical analysis for the same groups of rats and to select successfully denervated animals for immunohistochemistry and biochemistry. Extensive analysis on the functional effects of the striatal and

171

nigral lesions is presented in an accompanying paper (Herrera-Marschitz et al. 1986). Material and methods Male Sprague-Dawley (Pathogen-free; ALAB, Stockholm, Sweden) rats with free access to food and water ad libitum were used in all experiments. The rats were maintained in a temperature controlled environment on a 12 h light/dark cycle when not in experimental sessions.

Lesions Rats weighing 200-250 g were anaesthetized with a mixture of air and halothane and placed in a Kopf stereotaxic frame. With the skull oriented according to the K6nig and Klippel atlas (1963), ibotenic acid (IBA) injections (10.0 ~g/~l) were performed into the left striatum, using a Hamilton syringe with conically shaped cannula and a penetration tip of approximately 0.15 mm diameter, in six groups of rats. Other types of lesions were made into further three groups as described below. The coordinates were calculated from Bregma and the surface of the brain, but they are given according to the real stereotaxical targets, as described in the K6nig and Klippel atlas (1963). Group 1 (head) received 1.0 ~1 of IBA injected over a period of i min into the head of the striatum (A 9.7 mm, L -2.3 mm, V 0.5 ram). Group H (head/corpus) received two injections: 1.0 ~1 into the head of the striatum (coordinates: A 9.7, L -2.3, V 0.5) and 1.0 ~1 into the rostral part of the corpus of the striatum (coordinates: A 8.4, L -2.4, V 0.2). Group III (corpus/tail) received two injections: 1.6 ~1 into the corpus (coordinates: A 7.5, L -3.5, V -0.8) and 1.6 ~1 into the most rostral part of the tail (coordinates: A 6.9, L -2.8, V 0.2). Group IV (tail/corpus) received two injections, each of 1.6 ~1 volume into the tail and corpus of the striatum (coordinates: A 6.2, L -4.0, V 0.4 and A 7.2, L -2.2, V 0.4, respectively). Group V (tail/corpus) received three injections: 1.0 ~1 into the most caudal part of the tail (coordinates: A 4.9, L -4.1, V -0.4), followed by 1.0 gl into the middle of the tail (coordinates: A 6.2, L -2.6, V 1.4) and then 1.0 ~1 into the corpus (coordinates: A 7.5, L -2.0, V 0.4). Group VI (the whole striatum) received four IBA injections in the following order: 1.0 gl into the most caudal part of the tail (coordinates: A 4.9, L -4.4, V -0.6), 1.0 ~1 into the middle of the tail (coordinates: A 6.2, L -2.6, V 1.4), 1.0 gl into the corpus (coordinates: A 7.5, L -2.0, V 0.4) and then 1.0 gl into the head of the striatum (coordinates: A 8.5, L -2.3, V -0.4). Group VII (substantia nigra) (rats weighing 150-170 g) received 0.5 ~1 of IBA injection (10.0 ~g/~l) into the caudal part of the left substantia nigra, pars reticulata (SNR) (coordinates: A 1.8, L-2.0, V-2.6). Group VIII (medial forebrain bundle) (MFB) (rats weighing 150-170 g) received 4.0 gl of 6-hydroxy-DA (6OHDA) (2.0 ~g/91) into the left area ventralis tegmenti (coordinates: A 3.2, L -1.2, V-3.1). Finally, Group IX (lateral/medial striatnm) (rats weighing 200-250 g) received a single 1.0 ~tl IBA (10.0 gg/~l) injection either into the lateral (coordinates: A 6.7, L-4.0 and-4.3, V + 0) or medial (coordinates: A 7.7, L -2.0, V + 0) region of the corpus of the striatum. Behavioural evaluations - rotational behaviour In order to obtain a preliminary evaluation of the extent of the lesions produced by the unilateral intracerebral administration of

the various neurotoxins, the amount of behavioural asymmetric induced by these lesions was studied in a modified version of the original rotometer (Ungerstedt and Arbuthnott 1970) that allows the continuous recording of turns to the left or right with a detector utilizing infrared photocell barriers (Herrera-Marschitz and Ungerstedt 1984a). The IBA lesioned animals were treated with apomorphine 0.5 mg/kg s.c. 7-10 days after the lesion. Depending upon the extent of the lesion, this dose of apomorphine should induce ipsilateral rotation. The 6OHDA-treated animals were given apomorphine 0.05 mg/kg, s.c. two weeks after the lesion, which depending upon the extent of the lesion should cause them to rotate contralaterally (Ungerstedt 1971b). The rotational behaviour of each individual animal was expressed as the number of 360~ turn/rain for the entire duration of the behaviour (total rotation), or as turns/10 min (rotation intensity).

Dissection Rats were sacrificed by decapitation 10 to 20 days after the lesions. The nigral tissue was dissected from two adjacent sections through the midbrain, cut on a freezing microtome. The sampled tissue included the dorsal and lateral extent of the pars compacta and pars lateralis, respectively, and the crus cerebri adjacent to the pars reticulata. The tissue was weighed and immediately frozen on dry ice. The forebrain from each rat was fixed overnight in buffered formalin and stored in 10% sucrose for subsequent cresyl violet histology to characterize the extent of the lesion.

Immunohistochemistry Some animals from the various lesion groups (at least five animals of each) were processed for the immunohistochemical demonstration of the consequence of the lesion on nigral neurotransmitters and neuropeptides by the indirect immunofluorescence technique of Coons and collaborators (see Coons 1958; H6kfelt et al. 1973). Rats were anaesthetized with pentobarbital and perfused with icecold phosphate buffered normal saline followed by a chilled solution of 4% paraformaldehyde in 0.16 M sodium phosphate (pH 6.7) containing 0.3% picric acid (Zamboni and DeMartino 1967). After a six rain perfusion, the brain was removed and postfixed for 90 min in the same fixative. In some cases, an alternate fixation procedure (Pearse and Polak 1975; Lundberg et al. 1982) was used, allowing DYN peptides to be more readily visualized. Then, rats were anaesthetized and perfused as above. Brains were removed and 1-2 mm thick slices were cut and immersed in an ice cold solution of 0.25% parabenzoquinone and 2% para-formaldehyde in 0.05 M phosphate buffer for 90 min before transferring to a buffered sucrose solution. After at least 24 h in buffered sucrose, the brain was sectioned at a thickness of 14 ~m on a cryostat (Dittes, Heidelberg, FRG), and slide mounted sections were incubated with antisera raised against DYN (rabbit antiserum 84; see below), SP (rat monoelonal; Cuello et al. 1979, and polyclonal rabbit antiserum 497; see below), GAD (sheep polyclonal; Oertel et al. 1981a), tyrosine hydroxylase (TH) (rabbit polyclonal; Markey et al. 1980) or (Met) Enk (rabbit polyclonal 336; Schultzberg et al. 1979) overnight in a humid atmosphere at 4~ C. Antisera were diluted in phosphate buffered saline (PBS) containing 0.3% Triton X-100 (Hartman 1973). After rinsing in PBS, the primary antisera were visualized by incubation with fluoreseeinisothiocyanate (FITC)-labelled secondary antibodies to rabbit in the case of DYN, SP 497 and TH antisera, to rat in the case of monoclonal SP antiserum and to sheep in the case of GAD antiserum (Dakopatts, Copenhagen, Denmark; Amersham, Amersham, England; Miles Laboratories, Slough, England; American Qualex, La Mirada, California, USA; Sigma, St. Louis,

172 Table 1. Stepwise elution of a cation exchange column (SP Sephadex C-25), to separate peptides in the acetic acid extract of substantia nigra. Four ml of each buffer was used Buffer and fraction no

Concentration (M) pyridine: formic acid

I II

0.018 : 0.1 0,1 : 0.1

III

0.35 : 0.35

IV V

0.8 1.6

: 0.8 : 1.6

Peptide

(Met) Enk (Leu) Enk enkephalylhexapeptides SP, DYN B DYN A, DYN B

Missouri, USA). Sections were coverslipped with glycerol: PBS ( 3 : 1 ) containing p-phenylenediamine (Johnson, De Nogueira and Araujo 1981; Platt and Michael 1983) and examined in a Zeiss fluorescence microscope equipped with appropriate filters. For control the various antisera were absorbed with an excess of the respective peptide (5.0 or 25.0 ~tg peptide per ml antiserum diluted 1 : 100). Normal rabbit and sheep sera were used as controls for TH and GAD antisera, respectively.

separated by adding to the assay vial 200.0 ~ of a charcoal suspension consisting of 500.0 mg active charcoal and 50.0 mg dextran T 70 in 200.0 ml 0.05 M Na-phosphate buffer. The following antisera were used in the radioimmunoassays (cross-reactivity with other peptides are shown for each antiserum): DYN A: 84 +, with a final dilution of 1 : 500,000. Crossreactivity: DYN B, DYN A (1-8), DYN A (1-13), a-neoendorphin, (Leu) Enk, (Met) Enk and neurotensin < 0.1%. DYN A (9-17) = 100%. DYN B: 113 B at a final dilution of 1 : 500,000. Crossreactivity: as for 84 + with the exception of DYN A (9--17) and DYN A, which showed a cross-reactivity with 113 B < 0.1%. SP: 497, final dilution 1 : 100,000. Cross-reactivity: SP fragments 3-11 and 4-11 = 100%, 5-11 = 60%, 6-11 = 20%, substance K < 1%, SP 1-7, somatostatin, eledoisin, [3-endorphin, (Met) Enk and (Leu) Enk < 0.1%. (MeO Enk: 22, final dilution 1 : 800. Cross-reactivity: (Leu) Enk = 30%, (Met) Enk-Lys 6 = 25%, (Met) Enk-Arg 6 = 10%, Arg%(Leu) Enk = 4%, (Leu) Enk-Lys 6 and (Leu) Enk-Arg 6 < 1%, DYN A (1-8) < 0.01%. (Leu) Enk: 505, final dilution 1:3,000. Cross-reactivity: Arg~ Enk = 100%, (Met) Enk = 3%, DYN A, DYN A (1-8), (Met) Enk-Arg 6, (Met) Enk-Lys 6 and (Leu) Enk-Lys 6 < 0.1%.

HPLC Tissue extraction Tissues were homogenized with 1.0 ml 1 M acetic acid containing 0.01% mercaptoethanol. Samples were heated at 95~ C for five min, cooled on ice, homogenized by sordcation and then heated another five min at 95~ C. After cooling on ice, the samples were centrifuged for 10--15 rain in a Beckman Microfuge| and 0.8 ml aliquots of the supernatant were sampled.

Ion exchange chromatography A cation exchanger was used prior to the radioimmunoassay in order to concentrate and separate the different peptides in the extract. The method has been described in detail previously (Bergstrtm et al. 1983). Briefly, columns containing 1.0 ml of SP Sephadex C-25 were eluted in a stepwise manner with a mixture of pyridine and formic acid (Table 1). Two of the fractions were collected and assayed for immunoreactive peptides: fraction II containing (Met) and (Leu) Enk and fraction V (in this study fraction V also included fraction IV) containing DYN A, DYN B and SP. After the separation procedure, fractions were taken to dryness in a vacuum centrifuge and kept at -90 ~ C until further processing.

Radioimmunoassays The samples from the ion exchange procedure were dissolved in an appropriate volume of methanol: 0.1 M HCI (1 : 1), and 25.0 gl were incubated with antiserum and labelled peptide in the various radioimmunoassays. The radioimmunoassay procedure for DYN A and DYN B has been described elsewhere (Christensson-Nylander et al. 1985). The measurement of SP and the enkephalins followed a previously described radioimmunoassay procedure (Bergstrtm et al. 1983) with the following modifications: the assay buffer contained 0.05 M Na-phosphate, 0.82% NaC1, 0.93% Na-EDTA, 0.1% BSA and 0.1% gelatin, pH 7.4. The bound and free peptide were

HPLC identification was performed on a reversed phase column (RP-18, TSK-ODS-120-T, 5 ~t, Waters, USA) eluted with a gradient of 10% to 40% acetonitrile in 0.04% trifluoroacetic acid.

Chemicals DYN A, DYN B, (Met) Enk and (Leu) Enk were purchased from Bachem Feinchemikalien AG, Bnbendorf, Switzerland. SP was obtained from Peninsula Laboratories, San Carlos, CA, USA. SP Sephadex C-25 was purchased from Pharmacia Fine Chemicals, Uppsaia, Sweden. IBA (Sigma, St. Louis, USA) was dissolved in warm physiological Ringer solution. 6OHDA-HC1 (Sigma, St. Louis, USA) was dissolved in physiological saline with 0.2% ascorbic acid. Apomorphine HC1 (Apoteksbolaget, Stockholm, Sweden) was dissolved in warm physiological saline and injected s.c. into the left flank in a volume of 1.0 ml/kg body weight.

Statistics Means and standard error of the means (S. E. M.) were calculated and differences between means analysed with Student's t-test. Correlation coefficient was calculated using a least square method and the significance tested with F-ANOVA (F). A level of P < 0.05 for the one-tail test was considered as critical for statistical differences.

Results

Striatal I B A lesions (group I, 11, III, IV, V, l / l a n d IX) Behavioural analysis o f the lesions. I m m e d i a t e l y a f t e r the injection of IBA into the striaturn (groups I-VI), two different behavioural syndromes were observed.

173

Table 2. Rotational behaviour produced by a p o m o r p h i n e a in unilaterally lesioned rats N total (turns) Striatal lesions Group I 1 xlBA Group II 2xIBA Group IlI 2xlBA Group IV 2xIBA Group V 3 xIBA Group VI 4 x IBA Nigral lesions Group VII 1 xIBA(SNR) Group VIII 1 x 6 O H D A (MFB)

Ipsilateral rotation m a x i m u m intensity (turns/10 min)

14

47+7

10+2

6

21+7

total (turns)

Contralateral rotation m a x i m u m intensity (turns/10 min)

Duration (min) b

5 0 + 11

7+2

60+10

3+2

26+3

5+2

3 0 + 12

5

1 2 2 + 19

31+4

11+3

1+0.5

6 0 + 10

9

328+25

73+7

1 8 + 12

2+2

6 0 + 10

7

382+23

74+5

1+0.2

70+8

30

429 + 17

94 + 4

15 + 2

1 + 0.2

72 _+ 8

8

1 6 8 + 14

53+4

34+4

5+1

50+5

52

3 + 2

1 + 0.2

6+2

342 + 21

115 ___ 5

56 + 3

a IBA-lesioned rats were tested with a p o m o r p h i n e 0.5 mg/kg s.c. Rats lesioned with 6 O H D A injected into the left medial forebrain bundle were tested with a p o m o r p h i n e 0.05 mg/kg s.c. b total duration was calculated up to the last period showing 5 turns/10 min

When injected into the head and the corpus of the striatum, IBA induced a prolonged akinetic state, fall in rectal temperature and slowing of breathing. This state lasted several hours and led in some cases to death. This effect has been described previously (Schwarcz et al. 1979) as due to a prolongation of anaesthesia. However, IBA injected into the tail of the striatum produced an opposite effect, motor activation and awakening from the anaesthesia. We used this observation in order to reduce the high mortality otherwise produced by multiple IBA injections into the striatum. Injections were then performed in a caudo-rostral instead of a rostro-caudal order. This procedure significantly reduced acute mortality. Apomorphine (0.5 mg/kg s.c.) given one to two weeks after the lesion induced ipsilateral rotation which was closely correlated with the extent of the striatal lesions induced by IBA treatments, and also dependent upon the sites where the lesions were produced (Table 2). Apomorphine produced stronger rotational behaviour in animals receiving

IBA injections into the corpus and the tail than injections into the head and corpus of the striatum.

Cresyl violet-histological analysis of the lesion sites. The microscopic appearance of the striatum after the lesions with IBA was essentially as described by Schwarcz et al. (1979). The lesioned regions of the striatum were characterized by a virtually complete disappearance of neuronal somata and by a distinct gliosis. The boundaries of the lesion were often readily evident and only seldom was a border zone of shrunken, pyknotic neuronal cells noted. The extent of the lesion was relatively consistent with animals in a given group and the appearance of a typical lesion of the six main groups is shown in Fig. t. In a few cases, animals with misplaced lesions were noted in the histology. These animals invariably had aberrant values in the behavioural and biochemical data as well and were dropped from the analysis. In the following a brief description of the extent of the lesion in each of the six main groups is given: Group I (1 injection into the head of the striatum)

Pig. 1. Schematic illustration of extent of lesions of group I-VI as defined in Material and methods. T h e drawings are taken from the atlas of Paxinos and W a t s o n (1982) and represent frontal sections at the following levels: a = B r e g m a 2.2 m m ; b = B r e g m a 1.2 m m ; c = Bregma 0.2 m m ; d = B r e g m a -0.8 m m ; e = B r e g m a -1.8 ram; f = B r e g m a - 2 . 8 m m . T h e extent of a typical lesion in each group is shown by shaded areas. For further details see text. ace = nucleus accumbens; bst = bed nucleus of the stria terminalis; C C = corpus callosum; Co = commissura anterior; F = fornix; gb = globus pallidus; hi = hippocampus; IC = capsula interna; M F B = medial forebrain bundle; sep = septum; str = striatum; th = thalamus

#IFB ~F IB rlFB

~

MFB

e

....,~ ~,

MFB

J

~i

,~

l

f

>

Fig. 1

176

Fig. 2

177

was characterized by a loss of neurons in the most rostral part of the head of the striatum, in some cases extending into the claustrum and endopiriform nucleus (Fig. l-I). Group II (2 injections into the head and corpus of the striatum) showed an extensive lesion of the head of the striatum and a small impairment of the corpus and globus pallidus (Fig. 1-II). The medial-lateral extension of the lesion varied between animals and also the degree of extent into the fundus striati, as well as the effects on globus pallidus. Group III (2 injections into the corpus and tail of the striatum, large volume) had an almost complete lesion of the head and corpus of the striatum with partial, and between animals variable involvement of the globus pallidus (Fig. 1-III). In group IV (2 injections into the tail and corpus of the striatum, large volume) most of the head and corpus of the striatum as well as part of the tail were lesioned. Globus pallidus was only partially, but between animals somewhat variably affected (Fig. I-IV). The impairment of the corpus callosum, the claustrum and fundus striati varied between animals in groups III and IV. Group V (3 injections into the tail and corpus of the striatum) had a lesion of the entire head and corpus of the striatum and of the anterior half of the tail of the striatum; the globus pallidus and part of the entopeduncular nucleus were also lesioned (Fig. I-V). The deep cortical layers, specially dorsal, but also lateral to the corpus callosum were affected to a varying extent in the different animals, and this was true also for rats in group VI. Group VI (4 injections along the whole striatum) showed an extensive cell loss throughout both the head, corpus and the tail of the striatum, the globus pallidus and the entopeduncular nucleus (Fig. 1-VI). Some animals in this group also showed damage in the ventrolateral and reticular nuclei of the thalamus and/or the cortex, lateral to the striatum, as well as of anterior, lateral hippocampus. Some damage of the dorsal nuclear groups within the amygdaloid complex was also sometimes noted. A notion was that a thin zone of the periventricular region of the striatum as well as areas underlying the lateral aspects of the corpus callosum in many cases remained intact. In all cases the lesions spared the nucleus accumbens and, except as noted, the amyg-

daloid nuclei. These regions also contain neurons which are immunoreactive for dynorphin (Fallon et al. 1985; Vincent, Unpublished).

Immunohistochemical analysis of the substantia nigra. On the intact :side very dense networks of DYN (Fig. 2A; 3A), GAD (Fig. 3B, C), SP (Fig. 4E) immunoreactive fibers were observed in the SNR. Both GAD- and SP-like immunoreactivity (SP-LI) were fairly evenly distributed in the SNR, whereas the DYN-positive fibers seemed less densely packed in the mid-portion of the SNR. These very dense immunoreactivities were mainly localized in the SNR, but could sometimes, especially at rostral levels, be seen to extend into the zona compacta, alternating with moderately dense fiber networks immunoreactive to DYN (Fig. 2A), GAD (Fig. 3B) and SP (Fig. 4E). Dense (GAD) or moderately dense (DYN, SP) fiber networks were observed in the ventral tegmental area. (Met) Enk-LI exhibited a completely different distribution pattern with a moderately dense fiber network in the zona compacta (Fig. 4D; 5A) extending into the ventral tegmental area (Fig. 4A). Only very few positive fibers were seen in the SNR. TH-positive cell bodies were seen in the zona compacta (and at more caudal levels in addition in the SNR) with varicose dendrites extending into the SNR (Fig. 3D). After injection of IBA into the striatum (10-20 days), marked changes in DYN-, GAD- and SP-LI were observed in the substantia nigra, and these changes were correlated to the type of lesion. In general there was a parallel depletion of all three immunoreactivities (DYN, GAD and SP). Thus, although GAD often seemed less affected, the localization of the depleted areas within the SNR roughly overlapped for the three antisera. The most extensive effect was obtained with rats of group VI, i.e. after 4 injections of IBA. The "very dense'-type of DYNand SP-LI seen in the SNR and partly extending into the zona compacta disappeared almost completely, and only few fibers remained, localized mainly in the peripheral parts of the SNR, especially medially (Figs. 2B, 3E, 4B). In contrast, in the zona compacta a medium-dense fiber network of SP-positive fibers remained (cf. Fig. 4B, E). Also DYN-positive fibers

Fig. 2A, B. Immunofluorescence micrographs (montages) of the substantia nigra of rat of group VI, i.e. after four unilateral injections of ibotenic acid into the nucleus caudatus including its cauda. The micrographs are taken from the same section and shows control (A) and lesion (B) side after incubation with dynorphin (1-17) (DYN) antiserum. A very dense network of DYN-posifive fibers is Seen in the zona reticulata (zr) partly extending into the zona compacta (zc) (A). On the lesion side an almost complete disappeareance of DYN-positive fibers can be seen with only a few ones remaining in the zona compacta and in the pars lateralis (pl) of the substantia nigra. CC = crus cerebri; tob = tractus opticus basalis (accessory optic tract). Big arrows point dorsally, small arrows medially. Bar indicates 50 [xm. Both micrographs have the same magnification

00

179

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180

were still seen in the zona compacta. The DYN- and SP-positive fibers in the ventral tegmental area also seemed little affected by this lesion. In the same group (VI) some rats exhibited less complete effects, whereby dense networks of DYN- and SP-LI remained in the most medial and lateral parts of the SNR. Analysis of the extent of the lesion in such rats revealed that the periventricular area and the lateral peripheral zone of the striatum underlying the corpus callosum did not seem to be affected by the IBA injections. GAD-LI was also markedly reduced in the rats receiving 4 IBA injections, but a fairly dense network of fluorescent dot-like structures remained (Fig. 3F, G). Those GAD-positive fibers remaining after striatal lesions did not exhibit the classical "peridendritic arrays" described for GAD nerve endings in general in the zona reticulata by many authors (see Ribak et al. 1979; Mugnaini and Oertel, 1985), and as shown also in this paper. Striatal IBA injections did not seem to affect the number of THpositive cell bodies in the zona compacta or the dendritic processes extending into the SNR (Fig. 3H) at the time interval studied. No certain effect of striatal IBA injections could be observed on the number and distribution of (Met) Enk-immunoreactive fibers in the zona compacta (Figs. 4C, 5B) or in the ventral tegmental area (Fig. 4A). These figures are taken from a rat of the lesion group III (corpus/taft). Occasionally differences were encountered between lesion and control side in single sections (c.f. Fig. 4C, D), but this could not be observed in a sufficiently consistent way to allow statements on a lesion effect. Fig. 4C and D are adjacent to Fig. 4B and E, respectively, and allow a comparison of the strong depletion of SP-LI in the

SNR reticulata but lack of apparent effect on SP-LI and (Met) Enk-LI in the zona compacta. Less extensive depletions were seen in the substantia nigra of the groups of rats with lesions mainly affecting the head and the corpus of the striatum, leaving intact the lateral and often the medial regions. Again, overlapping depletion patterns were seen. In Fig. 4B, E the effect on SP-LI is demonstrated after a lesion of group III (corpus/tail). Results from experiments with IBA injections into medial or lateral halves of the corpus of the striatum (group IX) (not correlated with biochemistry) revealed a parallel depletion of DYN- (Fig. 5C), SP- (Fig. 5D), and GAD-LI (Fig. 5E) in the medial and lateral parts of the SNR, respectively (Fig. 6). Again, the depletion of GAD-LI was less pronounced (c.f. Fig. 5E with C, D), and after such lesions weakly fluorescent but distinct GAD-positive cell bodies could be seen in the zona reticulata (Fig. 5E). The medial (Fig. 6A-C) and lateral (Fig: 6D-F) localization, respectively, of the depletions and their extent in the rostro-caudal direction after the two types of IBA injections are indicated in Fig. 6. None of the staining patterns described above were seen after incubation with appropriate control sera.

Biochemical analysis of substantia nigra. The nigral levels of immunoreactive DYN A (ir-DYN A), irDYN B and ir-SP on the lesioned side compared to the control side, in the different groups of rats are shown in Table 3. A single injection with IBA into the rostral part of the head of the striatum (group I), gave no change

Fig. 3A-tI. Immunofluorescence micrographs of the substantia nigra of rat with unilateral ibotenic acid lesion of group VI (for further details, see Fig. 2) after incubation with antiserum to dynorphin (1-17) (DYN) (A, E), glutamic acid decarboxylase (GAD) (B, C, F, G) and tyrosine hydroxylase (TH) (D, H). A-D shows control side and E--H lesion side. A, B, D as well as E, F, H show two series of serial sections, respectively. A marked decrease in DYN-like immunoreactivity (LI) in zona reticulata (zr) can be seen, whereas fibers remain in zona compacta (zc) (c.f. A, E). A parallel disappearance of GAD-LI can be seen (c.f. B, F), but at higher magnifications numerous weakly fluorescent GAD-positive fibers can be seen in the zona reticulata of the lesion side. Note that the fibers do not exhibit a "peridendrific array" pattern after the lesion, but have a "dot-like" appearance (c.f. C, G). On the adjacent sections it can be seen that the dopamine (TH-positive) cell bodies in the zona compacta as well as their dendrites (big arrow heads) seem unaffected by the lesion (c.f. D, H). Small arrow heads in E, F, H point to the same structure to facilitate orientation. Bars indicate 50 ~tm. A, B, D, E, F, H have the same magnification. C, G have the same magnification Fig. 4A-E. Immunofluorescence micrographs of the ventral tegmental area (A), and medial substantia nigra (B-E) of a rat receiving two unilateral injections of ibotenic acid (group III) (corpus/tall) after incubation with antiserum to (Met) enkephalin (ENK) (A, C, D) and substance P (SP) (B, E). All micrographs have been taken from sections from the same rat and L indicates lesion side (A to the left, B, C) and C indicates control side (A to the right, D, E). B, C as well as D, E show adjacent sections, respectively. (A) In the ventral tegmental area (vta) dense ENK-positive networks are seen with a similar distribution and intensity both on the lesion (to the left) and control (to the right) side. Note very dense fiber network and cell bodies in the interpeduncular nucleus (ip). Asterisks indicate oculomotor nerve. (B-E) A marked depletion of SP-like immunoreactivity (LI) c a n be seen in the zona reticulata (zr) of the substantia nigra, whereas no certain difference is seen in the zona compaeta (zc) (c.f. B, E). The ENK-LI in the zona compacta on the adjacent sections is dense in both lesion and control side. In this particular pair of photographs the fiber network on the lesion side (C) appears somewhat less dense (c.f. C, D), but this is not a consistent finding. Bar indicates 50 ~tm. All micrographs have the same magnification

181

Fig. 5A-E. Immunofluorescence micrographs of the mid substantia nigra of sections from a rat of group III (A, B) and of a rat with a single injection of ibotenic acid into the lateral part of the corpus of the caudatus nucleus (C-E) (group IX) after incubation with antiserum to (Met) enkephalin (ENK) (A, B), dynorphin (1-17) (DYN) (C), substance P (SP) (D) and glutamic acid decarboxylase (GAD) (E). A, B are taken from the lateral part of the substantia nigra of the same section as shown in Fig. 4 (A shows control and B lesion side). C-E represent serial sections on the lesion side. (A, B) No certain differences can be seen in the distribution and intensity of ENK-immunoreactive fibers in the zona compacta (zc). Note that ENK-positive fibers are almost exclusively confined to the zona compacta with only single fibers in the zona reticulata (zr). (C-E) A well defined depletion of DYN-, SP- and GAD-like imnmnoreactivities can be seen in the lateral half of the zona reticulata. Note marked overlap of the depleted zones for all three immunoreactivities. However, numerous GAD-positive fibers remain, but these are of the punctuate type. Note GAD-positive cell bodies (arrows) which become visible after the lesion. The extent and localization of this lesion is shown schematically in Fig. 6D-F. CC = crus cerebri. Arrow in C points medially. Bar indicates 50 ~tm. All micrographs have the same magnification

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