In the present study we show that unilateral nerve transection induces changes of NOS expression and activity in frog Purkinje neurons, with differences in the time-course responses of the ipsilateral and contralateral sides of the corpus cerebelli. In general, the markers indicate a critical period of induction of NOS from 3 to 30 days after neurectomy, followed by down-regulation.
NOS Induction After Axotomy
Transection of a peripheral nerve branch results in a great degeneration of fibers and neurons (Lieberman, 1974; Donoff, 1995). However, some damaged neurons can survive if the injured nerve is able to regrow and successfully reinnervate the appropriate target, with the formation of functionally efficient synapses (Aguayo et al., 1991; Welcher et al., 1991). The regeneration capacity of CNS neurons is based on differences in their molecular responses to injury, and particularly on their ability to upregulate specific proteins for neuron survival. This ability may have potential as a therapeutic agent in the treatment of human neurodegenerative disorders (Lindsay, 1994; Vaudano et al., 1998).
There are molecules like NO which are particularly complex from a functional point of view (Prast and Philippu, 2001). In fact, NO is a biological signaling molecule that is important in the physiology and pathophysiology of many different tissues (Schulz et al., 1995). NADPHd and NOS protein are inducible in term of de novo synthesis in traumatically injured neurons (Wu, 2000). After peripheral axotomy, NOS protein and NOS mRNA increase in some neuronal populations (Verge et al., 1992; Vizzard et al., 1995); moreover, NOS is up-regulated in axotomized motoneurons (Yu, 1994; Yu, 1997; Müller and Stoll, 1998; Ma et al., 2000).
Both vulnerability and type of lesion have to be considered when induction of NADPHd or NOS is studied in vivo. In the mammal cerebellum, Purkinje cells normally do not contain NOS (Vincent, 1994; Blottner et al., 1995; Vincent, 1996), but a lesion can induce NADPHd in the perikaria (Blottner et al., 1995; Saxon and Beitz, 1996). Increased NOSi and NADPHd activity were described in dorsal root ganglia on the ipsilateral side to peripheral axotomy (Vizzard et al., 1995), from 3 days to 4 weeks. Similar results were obtained after electrolyte lesions of the nucleus basalis magnocellularis (Sabbatini et al., 1999). Unilateral ligation of L5 and L6 spinal nerves results in an ipsilateral increase of NOS-like immunoreactivity (Meller and Gebhart, 1993).
In our research we have taken into account that Purkinje cells are the only output of the cerebellar cortex, and therefore analysis of this type of neuron can represent the cerebellum's response to injury.
In the frog cerebellum (Rana perezi, Xenopus laevis) in normal conditions, absence of NOS activity in the Purkinje cell population, or weak positivity in some of them, has been observed (Brüning and Mayer, 1996; Muñoz et al., 1996; Alonso et al., 2000).
In our study, unilateral nerve transection induced NADPHd activity and NOS protein in Purkinje cells of Rana esculenta. While the control animals had less than 20% of weakly NADPHd-positive cells, 3 days after neurectomy 82% of cells became labeled on the ipsilateral side. An increased reactivity was evident at 15 days, when up-regulation of NOS enzymatic activity also occurred on the contralateral side. A similar trend was observed for NOS-immunoreactivity.
These findings demonstrate that the first important result in situ is that the Purkinje cells, i.e., the cerebellar neurons that play a role in motor coordination and learning (Ito, 1982), of the corpus cerebelli are involved in vestibular compensation. Most of the reports in the literature concern the involvement of the vestibular nucleus or the vestibulocerebellum, such as the flocculus (Kitahara et al., 1997, 1999; Zheng et al., 2001), in the compensation process. As regards more specific studies on the effects of vestibular neurectomy in the frog, since the early 1970s Dieringer has studied the vestibular nucleus, spinal cord, and cerebellum. Dieringer (1995) reported the sole finding on the response of Purkinje neurons to stimulation of the eighth nerve; however, no mention was made of the Purkinje cells of the corpus cerebelli or vestibulocerebellum.
The cytochemical changes we observed are explained by the presence of vestibular fiber projections in the Purkinje cell layer of the corpus cerebelli (Kuruvilla et al., 1985). The ascending branch of the ventral root of the eighth nerve sends primary fibers (mossy fibers) to the granule cell layer of the auricular lobe of the ipsilateral and contralateral sides and to the ipsilateral acoustic area of the brain stem, which, in turn, is connected to the contralateral acoustic area. The small number of thin fibers which terminate within the Purkinje cell layer of the corpus cerebelli could represent either collaterals of mossy fibers or a population of climbing fibers. Moreover, secondary vestibular fibers (climbing fibers) from vestibular nuclei are distributed in the granule cell layer of the lobus vestibularis and corpus cerebelli, and in the molecular layer of both sides, terminating on the Purkinje cell dendrites. In turn, Purkinje cells axons terminate on the cerebellar and statoacoustic nuclei (Gregory, 1974; Nieuwenhuys and Opdam, 1976; Kuruvilla et al., 1985; Montgomery, 1988; ten Donkelaar, 1998).
In the field of research on the effects of axotomy, the increased NO production in our model (at different times on the ipsilateral and contralateral sides) is also a new finding. This can be explained by the anatomical distribution of fiber projections in the frog cerebellum (see above), and in particular by the interconnections between the two vestibular nuclei. These projections suggest that the ipsilateral side to the lesion is altered earlier (3 days after neurectomy) and that only secondarily, via vestibular nuclei, does the contralateral area show changes (at 15 days). Our findings point out that changes of NOS expression in Purkinje cells of the frog cerebellum are indicative of the time-course and dynamics of vestibular compensation (Markham and Yagi, 1984).
Moreover, the pattern of markers of the contralateral side suggest that it is advisable to maintain caution in the use of this area as a control in experiments dealing with cellular processes of degeneration and regeneration.
NOS/NADPHd induction was observed at different times on the ipsilateral and contralateral sides in other models, such as in the rat lateral geniculate nucleus, after cauterization of limbus-draining veins (Wang et al., 2000).
NOS involvement in vestibular compensation has been widely investigated (for review, see Darlington and Smith, 2000). In a study of the frog, Flugel et al. (1994) were the first to find that inhibition of NOS prevents functional restoration after vestibular lesion. Kitahara et al. (1997) showed that changes in NOS expression occur in the rat cerebellar flocculus after unilateral vestibular deafferentiation, and that injection of L-NAME into the flocculus inhibits the compensation process. Kitahara et al. (1999) also reported that NOS expression in the flocculus modulates the expression of c-Fos in the vestibular nucleus complex following unilateral vestibular deafferentiation.
The importance of NOS in vestibular compensation was demonstrated in the current study by a comparison of the time-courses of behavioral changes and the cytochemical changes of the Purkinje cells. After neurectomy of the eighth nerve, the frogs, as expected (Goltz, 1870; Beritoff, 1928), assumed an asymmetrical posture that was maintained for about 3 days, after which the frogs recovered an almost normal posture (but only in static conditions). Normal behavior, in both static and dynamic conditions, was observed about 15 days after the unilateral vestibular neurectomy.
Finally, with regard to the recovery of cell functionality, 30-day-transected frogs showed a steady pattern in both the percentage and reactivity of Purkinje cells. A re-establishment of the normal pattern was achieved at 60 days, as shown by the intensity of NADPHd reaction but not NOSi, which remained in a high percentage of neurons (mainly on the ipsilateral side).
A down-regulation of NOS expression at late stages of axotomy or following mechanical lesions was recently reported in the spinal motoneurons after complete regeneration of lizard tail, although NADPHd and NOS reactivities persisted in the majority of the spinal motoneurons (Cristino et al., 2000).
However, our findings indicate a discrepancy between the two histochemical reactions. NOSi persisted in a large part of the Purkinje neurons 60 days after neurectomy, whereas NADPHd labeling did not. A possible interpretation of this result is that NOS protein is still higher than in controls, but it is inactive being NADPHd reactivity absent. The excess of NOS suggests that 60 days after transection the recovery is still in a critical phase that can be better evaluated together with other features of frog cerebellar architecture. Discrepancies between NOS expression and NOS activity/NADPHd labeling have been detected in the vestibular nucleus following unilateral labyrinthectomy (Zheng et al., 2001), as well as in other neurobiological models (Gabbott and Bacon, 1993; Cooke et al., 1994; Pisu et al., 1999). Possible reasons for these discrepancies are: 1) there are different isoforms of NOS responsible for NADPHd activity, 2) the NOS molecule contains an NADPH binding site that is inactive after fixation, or 3) the NOS molecule does not have an NADPH binding site. Furthermore, conformational changes of the molecule affecting the immunoreactive responses cannot be excluded.
What Is the Role of NO After Axotomy?
The gaseous free radical NO, which is generated in biological tissues, is an important regulator of a broad range of functions (Moncada et al., 1991; Alonso et al., 2000; Vincent, 2000) and is considered a factor in stimulating neurotransmitter release in the CNS (Ohkuma and Katsura, 2001). Regarding the CNS, research on different neurobiological models from vertebrates and invertebrates has produced fundamental findings on the role of NO in the normal physiology of several areas. It is known that NO is involved in the behavior of animals, i.e., feeding processes, visual functionality, memory, and learning (Chapman et al., 1992; Moroz et al., 1993; Elphick et al., 1995, 1996; Müller, 1996, 1997; Seidel and Bicker, 1997; Conforti et al., 1999; Pisu et al., 1999).
In the cerebellum NO is a marker of synaptic plasticity (Vincent, 1996; Alonso et al., 2000). However, NO has a critical and complex role in pathophysiology and response to injury. The most important question concerns the cytotoxic or neuroprotective role of NO when NOS induction occurs. It is likely that both roles for NO coexist in the same CNS region, even in the same neuronal population, at different times following injury. This could involve other chemical properties of the CNS region (Grassi and Pettorossi, 2001).
The induction of NOS a few days after nerve transection coincides with the first signs of the system's plasticity (i.e., degeneration process). After unilateral neurectomy, cytoskeletal phosphorylated neurofilaments in some Purkinje neurons' soma were observed (Bernocchi et al., 1994), in agreement with other researchers' findings concerning axonal injury or nerve crush (Sinicropi and McIlwain, 1983; Rosenfeld et al., 1987; Gold et al., 1991). In some cells degenerative changes may be irreversible and may result in cell death (Vignola et al., 1992), as shown by the lower density of the Purkinje cell layer starting from 15 days. This means that the appearance of NOS in Purkinje cells at early stages after neurectomy may be linked to a neurotoxic effect of the molecule.
Increased NOS activity was also detected in vestibular nuclei following labyrinthectomy (Zheng et al., 2001); nevertheless, degeneration of neurons in these nuclei was not observed. However, several neurochemical changes have been observed in these nuclei, such as modifications of GABA immunoreactivity in the cat (Tighilet and Lacour, 2001) and transient increase of NMDA receptor subunit expression in the guinea pig (Sans et al., 1997). Neurochemical changes were found not only in neurons but also in glial cells. During the recovery from injury to the peripheral vestibular system, GFAP-immunoreactivity changes in the astroglia of the lateral vestibular nuclei in the cat were shown (Cass and Goshgarian, 1990). Increased levels of astroglial S100 immunoreactivity were also found in guinea pig after labyrinthectomy (Rickmann et al., 1995). Data indicate a role of glial cells in neuroplasticity during vestibular compensation.
At present, the different degrees of response to injury of Purkinje cells and vestibular nuclei neurons are quite intriguing, and further experimental investigations are necessary.
In addition to the changes of Purkinje neurons, starting from 3 days after neurectomy, large-caliber fibers, which are very strongly NADPHd-reactive, were present in the granule cell layer. These look like degenerating fibers, as reported previously in the frog cerebellum (Bernocchi et al., 1994) and in other areas of the vertebrate CNS (Kaplan and Clemente, 1981; Marbey and Browner, 1987; Oestreicher et al., 1988).
The fiber distribution in the granule cell layer of 60-day-operated frogs was restored, and thin and beaded NADPHd-reactive axons were present. These regenerative signs support the idea that the persistence of NOS immunolabeling in Purkinje neurons at 2 months after neurectomy provides evidence for the neurotrophic or neuroprotective function of this molecule.
The factors that govern the effects of NO on cell physiology are complex and not completely understood. They appear to be related to cell type and NO concentration, and to the production of other free radicals or molecules that are also expressions of the functional involvement of the neural cells (Murphy et al., 1993; Schulz et al., 1995; Whittle, 1995; Vincent, 1996; Peuchen et al., 1997; Steinbusch et al., 2000).