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The effect of electrolytic thalamic lesions on the NADPH-diaphorase activity of neurons of the laterodorsal tegmental and pedunculopontine nuclei in rats
Journal of Chemical Neuroanatomy 17 (2000) 227 – 232 www.elsevier.com/locate/jchemneu
The effect of electrolytic thalamic lesions on the NADPH-diaphorase activity of neurons of the laterodorsal tegmental and pedunculopontine nuclei in rats Veronika Ne˘mcova´ a, Pavel Petrovicky´ a,*, Hans J. ten Donkelaar b a
Anatomical Institute of the First Medical Faculty, Charles Uni6ersity, U nemocnice 3, 12800 Prague 2, Czech Republic b Departments of Anatomy and Neurology, Uni6ersity of Nijmegen, Nijmegen, The Netherlands Received 23 August 1999; accepted 18 November 1999
Abstract Cholinergic neurons of the mesopontine complex have extensive ascending projections to the forebrain: the laterodorsal tegmental nucleus extensively innervates the anterior thalamus, the anteroventral nucleus in particular, whereas the pedunculopontine nucleus has widespread projections to both the thalamus and extrapyramidal structures. Most of their neurons express nitric oxide synthase (NOS) activity. Following electrolytic lesions of the anteroventral thalamic nucleus, nicotinamide adenine dinucleotide phosphate-diaphorase (NADPHd) activity in neurons of the laterodorsal tegmental nucleus changed drastically. The intensity of NADPH-diaphorase staining increased in laterodorsal tegmental neurons ipsilateral to the lesion side, but decreased contralaterally. The intensity of the NADPH-diaphorase staining of neurons of the pedunculopontine nucleus, however, remained unchanged bilaterally. After partial lesions of the anteroventral thalamic nucleus a similar effect was noted. In contrast, large electrolytic lesions involving other thalamic nuclei or extrapyramidal structures did not change the number of NADPH-diaphorase neurons or their intensity of staining in the laterodorsal tegmental nuclei. These data show that electrolytic lesions of target areas can lead to an upregulation of NOS expression in the parent cell bodies, provided that there is no wide collateralization as found for the pedunculopontine nucleus. © 2000 Elsevier Science B.V. All rights reserved. Keywords: Cholinergic neurons; Mesopontine cholinergic complex; Ascending projections; Nitric oxide; NADPH-diaphorase
1. Introduction The equivalence between the nicotinamide adenine dinucleotide phosphate-diaphorase (NADPHd) staining pattern and the nitric oxide synthase (NOS) distribution in the nervous system has been shown in aldehydefixed tissue (Bredt et al., 1991; Hope et al., 1991). Changes in the NADPHd/NOS expression of central and peripheral neurons or glial cells have been found following axotomy (Brecht et al., 1995; Ruan et al., 1995; Vizzard et al., 1995; Ando et al., 1996; He et al., 1996; Rossiter et al., 1996; Wu, 1996; Zhang et al., 1996; Zhao et al., 1996; Mariotti et al., 1997; Yick et al., 1998) or following inhibition of target structures (Mariotti and Bentivoglio 1996). Changes have also * Correspondindg author. Tel.: +420-2-24915003; fax: +420-2297692. E-mail address: [email protected] (P. Petrovicky´)
been found in various brain diseases, during stress or after endocrine changes (e.g. Ceccatelli et al., 1996; Chou et al., 1996; Hunot et al., 1996; Schenk et al., 1996; Woodside and Amir, 1996; Krukoff and Khalili, 1997). The cholinergic neurons of the mesopontine cholinergic complex (Butcher, 1995), i.e. the laterodorsal tegmental and pedunculopontine nuclei, are intensely stained with the NADPH-diaphorase (NADPHd) technique (Vincent et al., 1983; Ne˘mcova´ et al., 1997). Previous studies (Pape and Mager, 1992; Leonard et al., 1995; Leonard and Lydic, 1997) suggest that the nitric oxide released in such neurons does not act only in these nuclei, but also in their distant target structures. Cholinergic neurons of the mesopontine complex have extensive ascending projections reaching: (1) thalamic nuclei (anterior, reticular, mediodorsal, central medial, parafascicular, posterior, and habenular nuclei); (2) the
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lateral hypothalamus; (3) extrapyramidal structures (substantia nigra, subthalamic nucleus, entopeduncular nucleus, globus pallidus, and striatum); (4) the medial septum and the cingulate cortex, and (5) some brainstem structures (superior colliculus, pretectal area, and interpeduncular nucleus). The laterodorsal nucleus provides cholinergic projections preferentially to anterior thalamic regions and rostral portions of the basal forebrain, whereas the pedunculopontine nucleus predominantly innervates extrapyramidal structures (Sofroniew et al., 1985; Satoh and Fibiger, 1986; Woolf and Butcher, 1986; Hallanger and Wainer, 1988; Cornwall et al., 1990; Petrovicky´ et al., 1990; Butcher, 1995; Usunoff et al., 1999). The thalamic nuclei are densely innervated by NADPHd-positive fibers (Bertini and Bentivoglio, 1997; Kharazia et al., 1997). The aim of the present study was to examine the effect of electrolytic lesions in thalamic nuclei on the NOS expression of neurons of the mesopontine cholinergic complex and mamillary body with NADPHd histochemistry. Moreover, neurons of the laterodorsal tegmental nucleus were examined for retrograde cell changes.
2. Materials and methods A total of 49 adult Wistar rats of 300 g were used. In 37 rats, unilateral electrolytic lesions were made in the thalamus or neighbouring structures (as controls for larger thalamic lesions exceeding the limits of the thalamus). Two rats were sham-operated (craniotomy without a lesion), and ten rats were used for NADPHd-staining only. All surgical procedures were performed under Phenobarbital anaesthesia (0.2 mg/ 100 g bodyweight).
2.1. Electrolytic lesions Under Phenobarbital anaesthesia, the animals were placed in a stereotactic apparatus. A small hole was drilled in the skull and a needle electrode was brought into the thalamus using Swanson (1992) or Fifkova´ and Marsˇala (1967) coordinates. As a more practical system, Fifkova´ and Marsˇala’s coordinates were chosen (anterior thalamus: AP 1 – 1.5, ML 0.5 – 1.5, depth 4–6). Five to 10 s of electric current (0.5 – 2.5 mA, voltage 5 – 15 mV) caused small electrolytic lesions. Afterwards, the electrode was removed, the skin wound closed and the rats were allowed to survive for 14 days. In parallel series (in preparation) the same experiments were carried out with survival times ranging from 2 h till 32 days. The clearest changes in the intensity of the NADPHd reaction among cells in the same section were seen after 14 days. In all other survival times used, however, the changes were detactable too. After the
postoperative survival time, the rats were sacrificed under deep ether anaesthesia and perfused transcardially with 4% paraformaldehyde in phosphate buffer (pH 7.4). The brains were quickly removed, cut into 0.5 cm slices and stored overnight in the same fixative. The following day the slices were placed into a 30% sucrose solution in phosphate buffer for cryoprotection. After 3–4 days the slices were cut into 40 m frontal sections on a freezing microtome. The sections were used for NADPHd histochemistry and cresylviolet staining. The location and the extent of the electrolytic lesions were studied in the cresylviolet-stained sections. Based on the structures damaged, the operated rats were divided into several groups: (1) animals (n = 10) with a lesion of the anteroventral thalamic nucleus; (2) animals (n = 18) with a lesion of other thalamic nuclei, and (3) control animals (n= 9) with lesions exceeding the thalamic borders.
2.2. NADPH-diaphorase histochemistry and cresyl6iolet staining For the demonstration of NADPHd activity a modified Scherer-Singler (Scherer-Singler et al., 1983) method was used (Ne˘mcova´ et al., 1997). For free-floating sections, half the concentrations of tetrazolium dye and beta-NADPH were enough. Our modification allowed a subdivision of NADPHd-positive neurons into dark blue Golgi-like cells, light blue Golgi-like cells with a distinguishable nucleus, Nissl-like cells, and various types of cell processes and NADPHd-positive fibers (see Petrovicky´ and Ne˘mcova´, 1995; Petrovicky´ et al., 1998).
3. Results The electrolytic lesions made in the present study resulted in various changes in NOS activity in the nuclei of the mesopontine cholinergic complex. Changes were found only in the laterodorsal tegmental nuclei, but not in the pedunculopontine nuclei. There were no changes in the number or intensity of staining of NADPHd-positive neurons in the neighbourhood of the lesions.
3.1. Complete contralateral decrease in the intensity of NADPHd staining in the laterodorsal tegmental nucleus 6ersus symmetry of staining of the pedunculopontine nucleus After large electrolytic lesions involving the anteroventral thalamic nucleus or the rostral part of the mamillothalamic tract, well-stained NADPHd-positive neurons were found in the laterodorsal nucleus on the side of the lesion. Contralateral to the lesion side, the
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NADPHd-activity of laterodorsal tegmental neurons was lower and no cell processes were labeled (Figs. 1A, 2A, B). The number of NADPHd-positive cells was the same on both sides. In contrast, the NADPHd-positive neurons in both pedunculopontine nuclei were well stained. In cresylviolet stained sections of the laterodorsal nuclei no evidence for retrograde cell degeneration such as cell swelling and chromatolysis was observed.
3.2. Partial contralateral decrease in the intensity of NADPHd staining of the laterodorsal tegmental nucleus 6ersus symmetry of staining of the pedunculopontine nucleus After small, partial lesions of the anteroventral thalamic nucleus, numerous slightly stained NADPHd-positive neurons were observed in the contralateral laterodorsal tegmental nucleus similar to those after large electrolytic lesions. But, additionally, some wellstained NADPHd-positive cells were found in the medial process of the laterodorsal nucleus contralateral to the lesion side (Figs. 1B, 2C, D). Ipsilateral to the lesion, NADPHd-positive neurons in the laterodorsal tegmental nucleus were well stained with the exception of some weakly stained cells in its ventral process. Again, after these small lesions the NADPHd-positive neurons of both pedunculopontine nuclei were well stained.
Fig. 1. Diagrammatic representation of the distribution of NADPHdiaphorase stained cells in the laterodorsal tegmental nucleus: A, after a complete lesion of the right anteroventral thalamic nucleus; B, after a partial lesion of the right anteroventral thalamic nucleus. Filled dots represent deeply stained neurons, and open dots slightly stained neurons without stainable processes. Abbreviations: G, dorsal tegmental nucleus of Gudden; IC, inferior colliculus; mlf, medial longitudinal fascicle; R, rostral raphe nucleus.
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3.3. Changes in the intensity of NADPHd staining of neurons of the laterodorsal and pedunculopontine nuclei in experiments with lesions of other thalamic nuclei and surrounding structures No changes in the intensity of NADPHd staining were found in neurons of both laterodorsal tegmental and pedunculopontine nuclei after electrolytic lesions in other thalamic nuclei (ventral anterolateral complex, mediodorsal nucleus, intralaminar nuclei, parafascicular nucleus or posterior nuclei). Moreover, no changes in the intensity of NADPHd staining were observed in neurons of both laterodorsal tegmental and pedunculopontine nuclei after electrolytic lesions of structures surrounding the thalamus (striatum, entopeduncular nucleus, and corpus callosum). In animals with lateral hypothalamic lesions, a decrease of NADPHd-positivity was observed in the contralateral laterodorsal tegmental nucleus. Changes in NADPHd staining were similar to those after large electrolytic lesions. An increase of NADPHd expression was found in neurons of the ipsilateral medial nucleus of the corpus mamillare after electrolytic lesions of the mamillothalamic tract, and after lesions of the anteromedial thalamic nucleus (Fig. 2E, F).
4. Discussion Changes in the expression of NOS/NADPHd of neurons after axotomy are not unequivocal. After the electrolytic lesions of thalamic nuclei the following changes in NOS expression/NADPHd-positivity in neurons of the laterodorsal tegmental and pedunculopontine nuclei could be expected: (1) an increase of NOS expression like after axotomy of spinal ganglion cells and of cerebellar neurons (Saxon and Beitz, 1994); (2) NADPHd-positivity in normally unstained neurons such as appears during the degeneration of vagal motoneurons (Kristensson et al., 1995); (3) a decrease of NADPHd staining to basic levels such as found during the degeneration of dopaminergic mesencephalic neurons (Herdegen et al., 1993; Gonza´lez-Herna´ndez et al., 1997); (4) changes in the intracellular distribution of NADPHd such as found in the superior colliculus after eye enucleation (Tenorio et al., 1998), and (5) no changes in the number and intensity of staining of NADPHd-positive neurons at all. In the present study the number of NADPHd-positive neurons in the laterodorsal tegmental nuclei did not decrease until 14 days after the electrolytic lesions. Moreover, in cresylviolet stained sections no evidence for retrograde cell degeneration was found. The possibility that cell degeneration becomes evident later can not be excluded, however. After electrolytic lesions of axonal terminations in the anteroventral thalamic nu-
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Fig. 2. A – D: NADPH-diaphorase staining in the laterodorsal tegmental nucleus after a complete (A, ipsilateral to the lesion; B, contralateral side) and an incomplete (C, ipsilateral side; D, contralateral side) electrolytic lesion of the right anteroventral thalamic nucleus. E, F:NADPH-diaphorase staining in the medial mamillary nucleus after an electrolesion of the right mamillothalamic tract just behind its termination in the anterior thalamus (E, ipsilateral to the lesion; F, contralateral side). Magnification bar: 50 mm.
cleus we observed an increase of the intensity of NADPHd staining, i.e. an increase of NOS expression in the ipsilateral laterodorsal tegmental nucleus, and a decrease of the intensity of NADPHd staining, i.e. a decrease of NOS expression in the contralateral laterodorsal nucleus. These changes were dependent on the extent of the lesion in the anteroventral thalamic nucleus. The increase of NOS expression is most likely due to the axonal damage caused by the electrolytic
lesion. It should be emphasized that not all NADPHdpositive neurons of the laterodorsal tegmental nucleus project to the anterior thalamus. After the anterior thalamic lesion, however, all NADPHd-positive neurons in the laterodorsal nucleus showed an increase in NADPHd staining. This indicates that after axotomy NOS expression in neurons of the laterodorsal tegmental nucleus increases not only in damaged cells but also in the neighbouring neurons.
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The relatively weak, i.e. reduced NADPHd staining of neurons in the laterodorsal nucleus contralateral to the lesion side is unusual. Tract-tracing studies (Sofroniew et al., 1985; Satoh and Fibiger, 1986; Woolf and Butcher, 1986; Hallanger and Wainer, 1988; Cornwall et al., 1990) suggested that the cholinergic, NADPHd-positive neurons of the laterodorsal tegmental nucleus project largely ipsilaterally to the anterior thalamus. Contralateral thalamic projections from the laterodorsal tegmental nucleus appear to arise mostly from small, GABAergic neurons (Cornwall et al., 1990). Using HRP as a retrograde tracer, Petrovicky´ et al. (1990), however, showed that about one-third of the thalamic projections from the laterodorsal tegmental nucleus is aimed at the contralateral anteroventral, intralaminar and mediodorsal nuclei. Moreover, the thalamic projections from the laterodorsal nucleus show a relatively dense pattern of collateralization. Possibly, the axotomized cholinergic neurons of the laterodorsal nucleus with an increased NOS expression produce so much nitric oxide that they are able to inhibit the NOS expression/NADPHd-positivity of the contralateral laterodorsal tegmental nucleus. On the other hand, the small GABAergic laterodorsal neurons with their contralateral thalamic projections may increase their inhibition of the NADPHd-positive neurons in the contralateral laterodorsal nucleus. The absence of changes in the NOS expression of neurons of the laterodorsal tegmental nucleus after electrolytic lesions in thalamic nuclei other than the anteroventral nucleus may be explained by the predominance of the projections of the laterodorsal nucleus to the anteroventral thalamic nucleus. The absence of changes in the NADPHd-positivity of neurons of the pedunculopontine nuclei in all our experiments may be due to a wide pattern of collateralization within the diencephalon (Petrovicky´ et al., 1990), but moreover to its predominantly extrapyramidal projections. Even after basal ganglia lesions (in our control group) no changes were observed in the pedunculopontine nucleus. Apparently, destruction of only a limited number of axonal collaterals does not result in an increase of NOS expression detectable with NADPHd histochemistry. This is supported by our observations that hypothalamic and mamillothalamic tract lesions result in an increase of NOS expression in neurons of the laterodorsal tegmental nucleus. Ascending projections from the laterodorsal nucleus run together with the mamillothalamic tract through the hypothalamus (Woolf and Butcher, 1986; Petrovicky´ et al., 1990; Hayakawa et al., 1993). The increased intensity of NADPHd staining in the medial mamillary nucleus after lesioning its fiber tract or its main thalamic target is also in line with this notion. In conclusion, electrolytic lesions of the anteroventral thalamic nucleus lead to an upregulation of the NOS
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expression in cholinergic neurons of the laterodorsal tegmental nucleus, the target area for most of its NADPHd-positive neurons. In striking contrast, after such lesions as well as after lesions involving other thalamic nuclei or lesions of surrounding structures no changes in the NOS expression of the NADPHd-positive neurons of the pedunculopontine nucleus were found. This is probably due to the more widespread collateralization of pedunculopontine axons.
Acknowledgements The present study was supported in part by an ENP Short-term fellowship to Veronika Neˇmcova´ and by the Grant Agency of the Charles University (139/97) in the Czech Republic. The authors would like to thank K. Patkova´ and R. Kobianova´ for their technical assistance during the course of this study.
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The effect of electrolytic thalamic lesions on the NADPH-diaphorase activity of neurons of the laterodorsal tegmental and pedunculopontine nuclei in rats Veronika Ne˘mcova´ a, Pavel Petrovicky´ a,*, Hans J. ten Donkelaar b a
Anatomical Institute of the First Medical Faculty, Charles Uni6ersity, U nemocnice 3, 12800 Prague 2, Czech Republic b Departments of Anatomy and Neurology, Uni6ersity of Nijmegen, Nijmegen, The Netherlands Received 23 August 1999; accepted 18 November 1999
Abstract Cholinergic neurons of the mesopontine complex have extensive ascending projections to the forebrain: the laterodorsal tegmental nucleus extensively innervates the anterior thalamus, the anteroventral nucleus in particular, whereas the pedunculopontine nucleus has widespread projections to both the thalamus and extrapyramidal structures. Most of their neurons express nitric oxide synthase (NOS) activity. Following electrolytic lesions of the anteroventral thalamic nucleus, nicotinamide adenine dinucleotide phosphate-diaphorase (NADPHd) activity in neurons of the laterodorsal tegmental nucleus changed drastically. The intensity of NADPH-diaphorase staining increased in laterodorsal tegmental neurons ipsilateral to the lesion side, but decreased contralaterally. The intensity of the NADPH-diaphorase staining of neurons of the pedunculopontine nucleus, however, remained unchanged bilaterally. After partial lesions of the anteroventral thalamic nucleus a similar effect was noted. In contrast, large electrolytic lesions involving other thalamic nuclei or extrapyramidal structures did not change the number of NADPH-diaphorase neurons or their intensity of staining in the laterodorsal tegmental nuclei. These data show that electrolytic lesions of target areas can lead to an upregulation of NOS expression in the parent cell bodies, provided that there is no wide collateralization as found for the pedunculopontine nucleus. © 2000 Elsevier Science B.V. All rights reserved. Keywords: Cholinergic neurons; Mesopontine cholinergic complex; Ascending projections; Nitric oxide; NADPH-diaphorase
1. Introduction The equivalence between the nicotinamide adenine dinucleotide phosphate-diaphorase (NADPHd) staining pattern and the nitric oxide synthase (NOS) distribution in the nervous system has been shown in aldehydefixed tissue (Bredt et al., 1991; Hope et al., 1991). Changes in the NADPHd/NOS expression of central and peripheral neurons or glial cells have been found following axotomy (Brecht et al., 1995; Ruan et al., 1995; Vizzard et al., 1995; Ando et al., 1996; He et al., 1996; Rossiter et al., 1996; Wu, 1996; Zhang et al., 1996; Zhao et al., 1996; Mariotti et al., 1997; Yick et al., 1998) or following inhibition of target structures (Mariotti and Bentivoglio 1996). Changes have also * Correspondindg author. Tel.: +420-2-24915003; fax: +420-2297692. E-mail address: [email protected] (P. Petrovicky´)
been found in various brain diseases, during stress or after endocrine changes (e.g. Ceccatelli et al., 1996; Chou et al., 1996; Hunot et al., 1996; Schenk et al., 1996; Woodside and Amir, 1996; Krukoff and Khalili, 1997). The cholinergic neurons of the mesopontine cholinergic complex (Butcher, 1995), i.e. the laterodorsal tegmental and pedunculopontine nuclei, are intensely stained with the NADPH-diaphorase (NADPHd) technique (Vincent et al., 1983; Ne˘mcova´ et al., 1997). Previous studies (Pape and Mager, 1992; Leonard et al., 1995; Leonard and Lydic, 1997) suggest that the nitric oxide released in such neurons does not act only in these nuclei, but also in their distant target structures. Cholinergic neurons of the mesopontine complex have extensive ascending projections reaching: (1) thalamic nuclei (anterior, reticular, mediodorsal, central medial, parafascicular, posterior, and habenular nuclei); (2) the
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lateral hypothalamus; (3) extrapyramidal structures (substantia nigra, subthalamic nucleus, entopeduncular nucleus, globus pallidus, and striatum); (4) the medial septum and the cingulate cortex, and (5) some brainstem structures (superior colliculus, pretectal area, and interpeduncular nucleus). The laterodorsal nucleus provides cholinergic projections preferentially to anterior thalamic regions and rostral portions of the basal forebrain, whereas the pedunculopontine nucleus predominantly innervates extrapyramidal structures (Sofroniew et al., 1985; Satoh and Fibiger, 1986; Woolf and Butcher, 1986; Hallanger and Wainer, 1988; Cornwall et al., 1990; Petrovicky´ et al., 1990; Butcher, 1995; Usunoff et al., 1999). The thalamic nuclei are densely innervated by NADPHd-positive fibers (Bertini and Bentivoglio, 1997; Kharazia et al., 1997). The aim of the present study was to examine the effect of electrolytic lesions in thalamic nuclei on the NOS expression of neurons of the mesopontine cholinergic complex and mamillary body with NADPHd histochemistry. Moreover, neurons of the laterodorsal tegmental nucleus were examined for retrograde cell changes.
2. Materials and methods A total of 49 adult Wistar rats of 300 g were used. In 37 rats, unilateral electrolytic lesions were made in the thalamus or neighbouring structures (as controls for larger thalamic lesions exceeding the limits of the thalamus). Two rats were sham-operated (craniotomy without a lesion), and ten rats were used for NADPHd-staining only. All surgical procedures were performed under Phenobarbital anaesthesia (0.2 mg/ 100 g bodyweight).
2.1. Electrolytic lesions Under Phenobarbital anaesthesia, the animals were placed in a stereotactic apparatus. A small hole was drilled in the skull and a needle electrode was brought into the thalamus using Swanson (1992) or Fifkova´ and Marsˇala (1967) coordinates. As a more practical system, Fifkova´ and Marsˇala’s coordinates were chosen (anterior thalamus: AP 1 – 1.5, ML 0.5 – 1.5, depth 4–6). Five to 10 s of electric current (0.5 – 2.5 mA, voltage 5 – 15 mV) caused small electrolytic lesions. Afterwards, the electrode was removed, the skin wound closed and the rats were allowed to survive for 14 days. In parallel series (in preparation) the same experiments were carried out with survival times ranging from 2 h till 32 days. The clearest changes in the intensity of the NADPHd reaction among cells in the same section were seen after 14 days. In all other survival times used, however, the changes were detactable too. After the
postoperative survival time, the rats were sacrificed under deep ether anaesthesia and perfused transcardially with 4% paraformaldehyde in phosphate buffer (pH 7.4). The brains were quickly removed, cut into 0.5 cm slices and stored overnight in the same fixative. The following day the slices were placed into a 30% sucrose solution in phosphate buffer for cryoprotection. After 3–4 days the slices were cut into 40 m frontal sections on a freezing microtome. The sections were used for NADPHd histochemistry and cresylviolet staining. The location and the extent of the electrolytic lesions were studied in the cresylviolet-stained sections. Based on the structures damaged, the operated rats were divided into several groups: (1) animals (n = 10) with a lesion of the anteroventral thalamic nucleus; (2) animals (n = 18) with a lesion of other thalamic nuclei, and (3) control animals (n= 9) with lesions exceeding the thalamic borders.
2.2. NADPH-diaphorase histochemistry and cresyl6iolet staining For the demonstration of NADPHd activity a modified Scherer-Singler (Scherer-Singler et al., 1983) method was used (Ne˘mcova´ et al., 1997). For free-floating sections, half the concentrations of tetrazolium dye and beta-NADPH were enough. Our modification allowed a subdivision of NADPHd-positive neurons into dark blue Golgi-like cells, light blue Golgi-like cells with a distinguishable nucleus, Nissl-like cells, and various types of cell processes and NADPHd-positive fibers (see Petrovicky´ and Ne˘mcova´, 1995; Petrovicky´ et al., 1998).
3. Results The electrolytic lesions made in the present study resulted in various changes in NOS activity in the nuclei of the mesopontine cholinergic complex. Changes were found only in the laterodorsal tegmental nuclei, but not in the pedunculopontine nuclei. There were no changes in the number or intensity of staining of NADPHd-positive neurons in the neighbourhood of the lesions.
3.1. Complete contralateral decrease in the intensity of NADPHd staining in the laterodorsal tegmental nucleus 6ersus symmetry of staining of the pedunculopontine nucleus After large electrolytic lesions involving the anteroventral thalamic nucleus or the rostral part of the mamillothalamic tract, well-stained NADPHd-positive neurons were found in the laterodorsal nucleus on the side of the lesion. Contralateral to the lesion side, the
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NADPHd-activity of laterodorsal tegmental neurons was lower and no cell processes were labeled (Figs. 1A, 2A, B). The number of NADPHd-positive cells was the same on both sides. In contrast, the NADPHd-positive neurons in both pedunculopontine nuclei were well stained. In cresylviolet stained sections of the laterodorsal nuclei no evidence for retrograde cell degeneration such as cell swelling and chromatolysis was observed.
3.2. Partial contralateral decrease in the intensity of NADPHd staining of the laterodorsal tegmental nucleus 6ersus symmetry of staining of the pedunculopontine nucleus After small, partial lesions of the anteroventral thalamic nucleus, numerous slightly stained NADPHd-positive neurons were observed in the contralateral laterodorsal tegmental nucleus similar to those after large electrolytic lesions. But, additionally, some wellstained NADPHd-positive cells were found in the medial process of the laterodorsal nucleus contralateral to the lesion side (Figs. 1B, 2C, D). Ipsilateral to the lesion, NADPHd-positive neurons in the laterodorsal tegmental nucleus were well stained with the exception of some weakly stained cells in its ventral process. Again, after these small lesions the NADPHd-positive neurons of both pedunculopontine nuclei were well stained.
Fig. 1. Diagrammatic representation of the distribution of NADPHdiaphorase stained cells in the laterodorsal tegmental nucleus: A, after a complete lesion of the right anteroventral thalamic nucleus; B, after a partial lesion of the right anteroventral thalamic nucleus. Filled dots represent deeply stained neurons, and open dots slightly stained neurons without stainable processes. Abbreviations: G, dorsal tegmental nucleus of Gudden; IC, inferior colliculus; mlf, medial longitudinal fascicle; R, rostral raphe nucleus.
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3.3. Changes in the intensity of NADPHd staining of neurons of the laterodorsal and pedunculopontine nuclei in experiments with lesions of other thalamic nuclei and surrounding structures No changes in the intensity of NADPHd staining were found in neurons of both laterodorsal tegmental and pedunculopontine nuclei after electrolytic lesions in other thalamic nuclei (ventral anterolateral complex, mediodorsal nucleus, intralaminar nuclei, parafascicular nucleus or posterior nuclei). Moreover, no changes in the intensity of NADPHd staining were observed in neurons of both laterodorsal tegmental and pedunculopontine nuclei after electrolytic lesions of structures surrounding the thalamus (striatum, entopeduncular nucleus, and corpus callosum). In animals with lateral hypothalamic lesions, a decrease of NADPHd-positivity was observed in the contralateral laterodorsal tegmental nucleus. Changes in NADPHd staining were similar to those after large electrolytic lesions. An increase of NADPHd expression was found in neurons of the ipsilateral medial nucleus of the corpus mamillare after electrolytic lesions of the mamillothalamic tract, and after lesions of the anteromedial thalamic nucleus (Fig. 2E, F).
4. Discussion Changes in the expression of NOS/NADPHd of neurons after axotomy are not unequivocal. After the electrolytic lesions of thalamic nuclei the following changes in NOS expression/NADPHd-positivity in neurons of the laterodorsal tegmental and pedunculopontine nuclei could be expected: (1) an increase of NOS expression like after axotomy of spinal ganglion cells and of cerebellar neurons (Saxon and Beitz, 1994); (2) NADPHd-positivity in normally unstained neurons such as appears during the degeneration of vagal motoneurons (Kristensson et al., 1995); (3) a decrease of NADPHd staining to basic levels such as found during the degeneration of dopaminergic mesencephalic neurons (Herdegen et al., 1993; Gonza´lez-Herna´ndez et al., 1997); (4) changes in the intracellular distribution of NADPHd such as found in the superior colliculus after eye enucleation (Tenorio et al., 1998), and (5) no changes in the number and intensity of staining of NADPHd-positive neurons at all. In the present study the number of NADPHd-positive neurons in the laterodorsal tegmental nuclei did not decrease until 14 days after the electrolytic lesions. Moreover, in cresylviolet stained sections no evidence for retrograde cell degeneration was found. The possibility that cell degeneration becomes evident later can not be excluded, however. After electrolytic lesions of axonal terminations in the anteroventral thalamic nu-
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Fig. 2. A – D: NADPH-diaphorase staining in the laterodorsal tegmental nucleus after a complete (A, ipsilateral to the lesion; B, contralateral side) and an incomplete (C, ipsilateral side; D, contralateral side) electrolytic lesion of the right anteroventral thalamic nucleus. E, F:NADPH-diaphorase staining in the medial mamillary nucleus after an electrolesion of the right mamillothalamic tract just behind its termination in the anterior thalamus (E, ipsilateral to the lesion; F, contralateral side). Magnification bar: 50 mm.
cleus we observed an increase of the intensity of NADPHd staining, i.e. an increase of NOS expression in the ipsilateral laterodorsal tegmental nucleus, and a decrease of the intensity of NADPHd staining, i.e. a decrease of NOS expression in the contralateral laterodorsal nucleus. These changes were dependent on the extent of the lesion in the anteroventral thalamic nucleus. The increase of NOS expression is most likely due to the axonal damage caused by the electrolytic
lesion. It should be emphasized that not all NADPHdpositive neurons of the laterodorsal tegmental nucleus project to the anterior thalamus. After the anterior thalamic lesion, however, all NADPHd-positive neurons in the laterodorsal nucleus showed an increase in NADPHd staining. This indicates that after axotomy NOS expression in neurons of the laterodorsal tegmental nucleus increases not only in damaged cells but also in the neighbouring neurons.
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The relatively weak, i.e. reduced NADPHd staining of neurons in the laterodorsal nucleus contralateral to the lesion side is unusual. Tract-tracing studies (Sofroniew et al., 1985; Satoh and Fibiger, 1986; Woolf and Butcher, 1986; Hallanger and Wainer, 1988; Cornwall et al., 1990) suggested that the cholinergic, NADPHd-positive neurons of the laterodorsal tegmental nucleus project largely ipsilaterally to the anterior thalamus. Contralateral thalamic projections from the laterodorsal tegmental nucleus appear to arise mostly from small, GABAergic neurons (Cornwall et al., 1990). Using HRP as a retrograde tracer, Petrovicky´ et al. (1990), however, showed that about one-third of the thalamic projections from the laterodorsal tegmental nucleus is aimed at the contralateral anteroventral, intralaminar and mediodorsal nuclei. Moreover, the thalamic projections from the laterodorsal nucleus show a relatively dense pattern of collateralization. Possibly, the axotomized cholinergic neurons of the laterodorsal nucleus with an increased NOS expression produce so much nitric oxide that they are able to inhibit the NOS expression/NADPHd-positivity of the contralateral laterodorsal tegmental nucleus. On the other hand, the small GABAergic laterodorsal neurons with their contralateral thalamic projections may increase their inhibition of the NADPHd-positive neurons in the contralateral laterodorsal nucleus. The absence of changes in the NOS expression of neurons of the laterodorsal tegmental nucleus after electrolytic lesions in thalamic nuclei other than the anteroventral nucleus may be explained by the predominance of the projections of the laterodorsal nucleus to the anteroventral thalamic nucleus. The absence of changes in the NADPHd-positivity of neurons of the pedunculopontine nuclei in all our experiments may be due to a wide pattern of collateralization within the diencephalon (Petrovicky´ et al., 1990), but moreover to its predominantly extrapyramidal projections. Even after basal ganglia lesions (in our control group) no changes were observed in the pedunculopontine nucleus. Apparently, destruction of only a limited number of axonal collaterals does not result in an increase of NOS expression detectable with NADPHd histochemistry. This is supported by our observations that hypothalamic and mamillothalamic tract lesions result in an increase of NOS expression in neurons of the laterodorsal tegmental nucleus. Ascending projections from the laterodorsal nucleus run together with the mamillothalamic tract through the hypothalamus (Woolf and Butcher, 1986; Petrovicky´ et al., 1990; Hayakawa et al., 1993). The increased intensity of NADPHd staining in the medial mamillary nucleus after lesioning its fiber tract or its main thalamic target is also in line with this notion. In conclusion, electrolytic lesions of the anteroventral thalamic nucleus lead to an upregulation of the NOS
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expression in cholinergic neurons of the laterodorsal tegmental nucleus, the target area for most of its NADPHd-positive neurons. In striking contrast, after such lesions as well as after lesions involving other thalamic nuclei or lesions of surrounding structures no changes in the NOS expression of the NADPHd-positive neurons of the pedunculopontine nucleus were found. This is probably due to the more widespread collateralization of pedunculopontine axons.
Acknowledgements The present study was supported in part by an ENP Short-term fellowship to Veronika Neˇmcova´ and by the Grant Agency of the Charles University (139/97) in the Czech Republic. The authors would like to thank K. Patkova´ and R. Kobianova´ for their technical assistance during the course of this study.
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