The nucleus tegmenti pedunculopontinus and circling behaviour in the rat

Neuroscience Letters, 26 (1981) 11- 16

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Elsevier/North-Holland Scientific Publishers Ltd.

THE NUCLEUS TEGMENTI PEDUNCULOPONTINUS BEHAVIOUR IN T H E RAT

AND CIRCLING

1.C. KILPATRICK and M.S. STARR Department of Pharmacology, The School of Pharmacy, 29-39 Brunswick Square, London WC1N l A X (U.K.)

(Received April 16th, 1981; Revised version received June 8th, 1981; Accepted July 8th, 1981) Lesioning the rat's substantia nigra (SN) with kainic acid (0.8/~g) or by electrocoagulation (1 mA for 6 sec) significantly lowered GABA and glutamate decarboxylase levels at the treatment site, but not in the nucleus tegmenti pedunculopontinus (PPN), suggesting nigro-PPN fibres do not synthesize or store GABA. Stereotaxic injection of one PPN with muscimol (40 ng), picrotoxin (40 ng) or tetanus toxin (30 mouse LDs0 doses) had little or no effect on the animals' behaviour; kainate caused ipsilateral body flexion, sporadic ipsiversive circling and contraversive barrel-rolling. These behavioural abnormalities disappeared after 7 days when histology confirmed virtually complete loss of PPN perikarya, intense gliosis and some demyelination of passing axons. Impairing PPN transmission with kainate (chronically) or muscimol (acutely) caused weak apomorphine-induced contraversive rotation, but did not modify the robust nigral muscimol-evoked contraversive asymmetry. While we do not exclude a role of PPN in motor control, these data suggest that nigro-PPN neurones are neither GABAergic nor mediators of central dopaminergic function. Whilst it is recognized that a bilateral imbalance in nigrostriatal dopaminergic activity can lead to postural deviation or turning in circles (for review see ref. 22), the neural substrates o f this behaviour have only recently begun to emerge. In addition to the striatopallidal connection, attention has focussed on the importance o f the reciprocal striatonigral G A B A p a t h w a y in m o t o r control and has led to the hypothesis that the substantia nigra (SN) represents a m a j o r o u t f l o w station for basal ganglia influences on posture and l o c o m o t i o n [6,8,24]. O u r interest lies in identifying which o f the assorted efferent systems emerging f r o m the SN zona reticulata (SNR) is responsible for conveying such information. Detailed studies f r o m this l a b o r a t o r y have thus far implicated the m a j o r outputs to the ventromedial thalamus (VM) [14,16,19] and the superior colliculus (SC) [15] in both a p o m o r p h i n e - and nigral-induced circling in the rat. F r o m anatomical considerations, however, Wright and A r b u t h n o t t [25] and Graybiel and Ragsdale [10] have also advocated the m o r e modest projection f r o m the S N R to the nucleus tegmenti p e d u n c u l o p o n t i n u s ( P P N ) [1,4,10,11,24] as a likely mediator o f d o p a m i n e r g i c function, since, a l t h o u g h the terminations o f descending P P N efferents are as yet u n k n o w n , this nucleus could affect m o t o r behaviour t h r o u g h its projection to the SN z o n a c o m p a c t a [9,12] and by its reciprocal connections with the subthalamic and e n t o p e d u n c u l a r nuclei [2,9,10,12,21]. W e have therefore investigated the possibility that n i g r o - P P N neurones are instrumental in the 0304-3940/81/0000-O000/$02.50 © Elsevier/North-Holland Scientific Publishers Ltd.

12 execution of dopamine-dependent circling in the rat. Since the nigral efferents to VM and SC comprise inhibitory GABA-containing neurones [3,4,15,19,23], we have further considered whether nigro-PPN fibres, too, are GABAergic. Male Wistar albino rats (160-210 g) were anaesthetized with 1.5°70 halothane in oxygen and placed in a stereotaxic frame. Injections or lesions were made in SNR (A 1.75, L 2.0, D 2.9) or PPN (P 0.1-0.5, L i.0-1.4, D 1.3-1.6), coordinates being derived from KOnig and Klippel [17]. Drugs were dissolved in artificial cerebrospinal fluid (CSF) [23] and delivered in 0.2 gi over 3 min from a l gl Hamilton syringe. Anaesthesia was then discontinued and following rapid recovery (5-10 min) animals were placed in an open field in order to monitor the direction and intensity of induced asymmetries. Lesions were made either chemically by injecting kainic acid (0.8 #g), or electrically with a bipolar electrode (1 mA for 6 sec). All injection and lesion sites were examined histologically in l0 or 20 gm serial coronal brain sections stained with cresyl violet, phosphotungstic acid haematoxylin or by the Kl~ver-Barrera technique. Biochemical determinations of GABA content and of glutamate decarboxylase (GAD) activity were conducted on weighed samples of tissue dissected from brains that had been removed and frozen in solid CO2/acetone within 45 sec of death, by means of conventional microdansylation [23] and fluorimetric [18] procedures respectively. As can be seen from Table I, samples of PPN tissue collected from untreated control rats contained moderate amounts of GABA and its synthesising enzyme; GABA and GAD were also present in characteristically high levels in intact SN. Intranigral kainate injection caused, one week later, a massive though subtotal loss of neuronal perikarya within the sphere of injection (1 ram), which was confined to the centre of the caudal two-thirds of the SN zona reticulata (SNR) (for detailed histology see refs. 16 and 23). Electrocoagulation similarly damaged the SNR as well as some of the adjacent SN pars compacta and crus cerebri (typical lesion shown in ref. 16). The biochemical sequelae of these treatments were significant reductions in nigral GABA ( - 20.0 and -46.7o70) and GAD ( - 24.8 and -48.1070) for kainate and electrothermic lesions respectively (see Table I); neither treatment, however, altered GABA or GAD levels in the ipsilateral PPN 7-10 days later, suggesting that nigro-PPN fibres do not synthesize or store GABA (Table I). In order to assess whether the integrity of the PPN is essential for the normal maintenance of posture and/or locomotor activity, we attempted to selectively destroy PPN output neurones with a somatotoxin, kainic acid. Microinjection of 0.8/~g kainate (in 0.2 gl) into one PPN initiated tight, ipsilateral flexion of the neck and trunk lasting about 18 h, which was interspersed during the first 6 h by short bursts (10-30 sec) of tight ipsiversive circling (peak 28-42 turns/rain at 35 rain, n--8) and contralaterally-directed barrel-rolling. All rats then exhibited varying degrees of ipsilateral ataxia and flaccid dystonia of both hind limbs, coupled with a contraversive bias, resulting in slow, net forward ambulation that oscillated from right to left. These abnormalities of gait disappeared after 7 days, possibly through behavioural compensation, and are reminiscent of injury to the neighbouring

13 TABLE I EFFECTS OF KAINATE INJECTION OR ELECTROLESION OF THE SUBSTANTIA NIGRA (SN) ON GABA AND GLUTAMATE DECARBOXYLASE (GAD) LEVELS IN SN AND NUCLEUS TEGMENTI PEDUNCULOPONTINUS (PPN) Rats received an injection of 0.8 #g kainate or an electrolesion (1 mA for 6 sec) in the right-hand SN. They were killed 7-10 days later and both SN and PPN removed for assay of GABA content [20] and GAD activity [16]. Results are expressed as mean _+ S.E.M. * P<0.005, **P<0.001 by t-test. Nigral treatment

n

PPN

SN Left

Right

Left

Right

4.44_+0.14 4.71 _+0.11 4.48+-0.20

4.46_+0.18 4.53+-0.13 4.72+-0.24

68.7_+7.4 60.9_+5.5 68.0_+4.2

65.1_+8.1 59.3_+6.6 70.2_+4.9

G A B A concentration (#mol/g wet wt)

Untreated 5 Kainate 21 Electrolesion 6

7.92_+0.17 8.35+_0.40 8.42_+0.33

8.11_+0.20 6.68_+0.39* 4.49_+0.30**

G A D activity (#mol/g wet wt/h)

Untreated Kainate Electrolesion

6 6 5

122.2_+11.6 129.5_+1 0 . 2 133.0_+1 3 . 7

130.5_+9.2 97.4_+7.8* 69.0_+6.8**

s u p e r i o r cerebellar p e d u n c l e [7]. S u b s e q u e n t h i s t o l o g y o f the a r e a revealed virtually c o m p l e t e a b s e n c e o f P P N p e r i k a r y a a n d c o n s i d e r a b l e gliosis within a sphere of 0 . 3 - 1 . 0 m m d i a m e t e r , as s h o w n s c h e m a t i c a l l y in Fig. 1. W i t h i n the b r a c h i u m c o n j u n c t i v u m (BC) we o b s e r v e d evidence o f d e m y e l i n a t i o n o f s o m e fibres o f p a s s a g e at a d i s t a n c e o f up to 500/~m f r o m the needle track. H o w e v e r , this could reflect needle r a t h e r t h a n k a i n a t e d a m a g e , as it was equally c o m m o n in C S F injected rats a n d was a b s e n t in t w o rats w h o s e P P N sustained d a m a g e f r o m k a i n a t e d i f f u s i n g f r o m a n i n j e c t i o n sited i m m e d i a t e l y d o r s a l to a n d n o t p e n e t r a t i n g the BC. It is n o t e w o r t h y t h a t the ipsiversive circling episodes exhibited by these t w o animals were still as r a p i d a n d as tight, t h o u g h the incidence o f b a r r e l - r o l l i n g a n d later a t a x i a was greatly r e d u c e d . By c o n t r a s t , C S F - t r e a t e d rats a p p e a r e d o u t w a r d l y n o r m a l and d i d n o t b e c o m e ataxic. In fact gait d i s o r d e r s were severest with the m o s t c a u d a l k a i n a t e p l a c e m e n t s , p a r t i c u l a r l y where the i n j e c t i o n needle h a d p a s s e d t h r o u g h the BC a n d where, as a result, there was p r o n o u n c e d gliosis a n d r e d u c e d n e u r o n a l d e n s i t y in the s u b j a c e n t reticular f o r m a t i o n . In all cases the locus c o e r u l e u s and p a r a b r a c h i a l nucleus r e m a i n e d intact. C h a l l e n g i n g these k a i n a t e - l e s i o n e d rats with a p o m o r p h i n e (0.5 m g / k g s.c.) 24 h later elicited s h o r t b o u t s o f c o n t r a v e r s i v e r o t a t i o n s ( p e a k 9.3 _+ 1.8 t u r n s / m i n ) t h a t lasted a b o u t 35 min; this r e s p o n s e was w e a k e r at 60 h (7.3 +_ 1.1 t u r n s / r a i n ) and a b s e n t at 7 d a y s p o s t - l e s i o n , its d i s a p p e a r a n c e p a r a l l e l l i n g the a n i m a l s ' recovery from ataxia. T h e acute r e s p o n s e to u n i l a t e r a l P P N i n j e c t i o n o f m u s c i m o l (40 ng, n = 9) lasted 9 0 - 1 2 0 m i n a n d closely r e s e m b l e d that to c h r o n i c k a i n a t e ; i.e. ipsilateral a t a x i a a n d an u n d e r l y i n g c o n t r a v e r s i v e t e n d e n c y w h i c h , at 15 m i n , was c o n v e r t e d i n t o a c t u a l

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P 100

Fig. 1. Schematic coronal view of the rat's brain at the pontine level showing extent of neuronal loss and gliosis (stippled area) 7 days after an injection of kainic acid (0.8/zg in 0.2 ~1) centred in the nucleus tegmenti pedunculopontinus (ppn, approximate boundary indicated by dotted line), bc, brachium conjunctivum; cc, cerebral cortex; ic, inferior colliculus; pag, periacqueductal grey; rf, reticular formation; sc, superior colliculus. circling (peak 4 turns/min) by apomorphine. Similar injections into one P P N of picrotoxin (40 ng, n = 9), a G A B A antagonist, or of tetanus toxin (30 mouse LDs0 doses, n = 6), a c o m p o u n d which attenuates G A B A release [5], did not modify the rats' behaviour within 1 or 48 h, respectively. No asymmetries developed in 4 rats receiving a control injection of 0.2 #l CSF in P P N in one hemisphere and then challenged with apomorphine systemically. Since the striatonigral G A B A pathway is considered to be actively engaged in mediating a p o m o r p h i n e ' s effects on m o t o r behaviour [6,8,24], we also studied whether circling derived from stimulating nigral G A B A receptors could be altered by: (a) a kainate lesion, or (b) muscimol injection of the ipsilateral PPN. In control rats, unilateral intranigral injection of 40 ng muscimol (in 0.2 #l) provoked characteristic nose-to-tail contraversive turning (peak 22.5 +_ 1.4 turns/min, n--6). This response was elicited with equal facility in rats bearing a 7 day-old kainate lesion of the corresponding P P N (peak 21.4 _ 2.2 turns/min, n = 8). Similarly, if this circling behaviour was interrupted after 30 min, when the rats were averaging 21.8 _ 1.2 turns/min (n = 8), by briefly re-anaesthetizing the rats and injecting one half of the group with muscimol (40 rig) and the rest with CSF (controls) in the ipsilateral P P N , in either case a normal pattern of circling was resumed within 15-20 min (23.0 ± 1.9 and 20.6 ± 1.7 turns/min respectively). Moreover, under these conditions, muscimol delivered into the P P N did not produce ataxia, as though this phenomenon had been masked or suppressed by equivalent muscimol treatment of the SNR. These findings contrast with those involving other prospective nigral output centres in the VM and SC, where impairment of transmission with kainate or muscimol significantly attenuated nigral-derived muscimol turning, suggesting that these structures (unlike PPN) lie downstream of the SNR and mediate its effects on locomotor behaviour [14-16].

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Although the nigro-PPN projection was first described nearly 20 years ago [4L its physiology and function are still poorly understood. Earlier findings suggested this pathway was not dopaminergic, as it was resistant to intranigral 6-hydroxydopamine treatment [25]. Our own data argue against the idea that it might be GABAergic. Attempts to inject or lesion the PPN discretely, in order to evaluate its possible role in motor behaviour, are made difficult by its intimate proximity to a major fibre tract, the BC [1,4,10,25]. Even with control injections of CSF into the PPN there was some histological indication of perforant fibre degeneration, though this never achieved sufficient prominence in kainate-lesioned animals to suggest it was the principal cause of the unsteady gait (see ref. 7). In fact the ataxia appeared rather more closely related to loss of cells from the reticular formation immediately ventral to the caudal region of the PPN. Little is known of the motor function of this brain area, though recent work has emphasized the importance both of the dorsal mesencephalic reticular formation [20] and the neighbouring periacqueductal grey matter [13] as output centres for striatal influences on posture. We are less certain of the origin of the apomorphine-accelerated contraversive asymmetry in these experiments, since this contrast both with the ipsiversive circus movements previously attributed to superior cerebellar peduncular injury at the pontine level [7], and with those seen following similar impairment of transmission in VM or SC [14-16]. Should this circling prove to be related solely to PPN dysfunction, it could signify that the PPN has an influence (albeit weak) on motor activity which is opposite to that exercised by VM or SC. In either event, however, it seems unlikely that the signals governing this apomorphine-induced turning motion are routed via a striato-nigro-PPN circuit, in view of the complete insensitivity of nigral muscimol-evoked rotations to PPN manipulation. On the other hand, the PPN could mediate/modulate dopamine-dependent behaviours of this type via its known anatomical 'loop' associations with other basal ganglia nuclei (see ref. 10), or through its suspected descending connections with the reticulospinal circuitry of the lower brainstem [10,21]. These results are in broad agreement with the observations of Jenner et al. [13], who discovered that secondary lesions of the ipsilateral PPN did not modify the circling generated by dopamine agonists in rats bearing unilateral lesions of the nigrostriatal dopamine tract. To conclude, the experimental data currently available do not support the earlier suggestion [25] that nigro-PPN neurones may serve as an output route for the extrapyramidal motor system, though a more subtle (modulatory?) participation of PPN in motor behaviour cannot be excluded. We are grateful to the Medical Research Council for financial support. 1 Beckstead, R.M., Domesick, V.B. and Nauta, W.J.H., Efferent connections of the substantia nigra and ventral tegmental area in the rat, Brain Res., 175 (1979) 191-217. 2 Carter, D.A. and Fibiger, H.C., The projections of the entopeduncular nucleus and globus pallidus in the rat as demonstrated by autoradiography and horseradish peroxidase histochemistry, J. comp.

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