Sensory (nociceptive) stimulation evokes Fos expression in the subthalamus of hemiparkinsonian rats.

OBJECTIVE: In an effort to understand cell activity patterns and sensorimotor integration in Parkinson's disease, we have explored the expression of the Fos protein in the subthalamus after sensory (nociceptive) stimulation of hemiparkinsonian Sprague-Dawley rats [6-hydroxydopamine [6OHDA]-lesioned]. Fos is a marker for neuronal activity in most areas of the brain and the subthalamus is a major driving force of the basal ganglia and target for surgical intervention in parkinsonian patients. METHODS: The medial forebrain bundle (major tract carrying dopaminergic nigrostriatal axons) was injected with either 6OHDA or saline (controls). A week later, some rats were subjected to mechanical stimulation (pinching; activating nociceptive pathways) of the hindpaw for 2 hours, while others received no stimulation. Thereafter, brains were processed using routine tyrosine hydroxylase (TH; marker for dopaminergic cells) or Fos immunocytochemistry. RESULTS: In the cases that had saline injections combined with mechanical stimulation or with no stimulation, as well as those that had 6OHDA lesions combined with no stimulation, there were no Fos+ cells in the subthalamus. However, in the cases that had 6OHDA-lesions combined with mechanical stimulation, there were many Fos+ cells within the subthalamus of both sides, particularly on the ipsilateral side. DISCUSSION: Our results indicate that after an increase in sensory (nociceptive) activity, via mechanical stimulation, there is an induction of Fos expression in the subthalamus of 6OHDA-lesioned cases. We suggest that activating nociceptive pathways exacerbates the abnormal cell activity in the basal ganglia generated by the hemiparkinsonian condition.

Survival of midbrain dopaminergic cells after lesion or deep brain stimulation of the subthalamic nucleus in MPTP-treated monkeys.

We have examined dopaminergic cell survival after alteration of the subthalamic nucleus (STN) in methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated monkeys. The STN was lesioned with kainic acid (B series) or underwent deep brain stimulation (DBS) at high frequency (C series). In another series, MPTP-treated and non-MPTP-treated monkeys had no STN alteration (intact animals; A series). Animals were treated with MPTP either after (B1, C1) or before (B2, C2) STN alteration. We also explored the long-term ( approximately 7 months) effect of DBS in non-MPTP-treated monkeys (D series). Brains were aldehyde-fixed and processed for routine Nissl staining and tyrosine hydroxylase immunocytochemistry. Our results showed that there were significantly more (20-24%) dopaminergic cells in the substantia nigra pars compacta (SNc) of the MPTP-treated monkeys that had STN alteration, either with kainic acid lesion or DBS, compared to the non-MPTP-treated monkeys (intact animals). We suggest that this saving or neuroprotection was due to a reduction in glutamate excitotoxicity, as a result of the loss or reduction of the STN input to the SNc. Our results also showed that SNc cell number in the B1 and C1 series were very similar to those in the B2 and C2 series. In the cases that had long-term DBS of the STN (D series), there was no adverse impact on SNc cell number. In summary, these results indicated that STN alteration offered neuroprotection to dopaminergic cells that would normally die as part of the disease process.

Fos immunoreactivity in some locomotor neural centres of 6OHDA-lesioned rats.

In this study, we explore Fos expression (a measure of cell activity) in three nuclei associated with locomotion, namely the zona incerta, pedunculopontine tegmental nucleus and cuneiform nucleus (the latter two form the mesencephalic locomotor region) in hemiparkinsonian rats. Sprague-Dawley rats had small volumes of either saline (control) or 6 hydroxydopamine (6OHDA) injected into the medial forebrain bundle, the major tract carrying dopaminergic nigrostriatal axons. After various post-lesion survival periods, ranging from 2 h to 28 days, rats were perfused with formaldehyde and their brains processed for routine tyrosine hydroxylase and Fos immunocytochemistry. Our results showed a significant increase (P < 0.05) in the number of strongly labelled Fos+ cells in the cuneiform nucleus in the 6OHDA-lesioned cases compared to the controls after 7 and 28 days survival periods. By contrast, there were no significant differences (P > 0.05) in the number of strong-labelled Fos+ cells in the zona incerta and pedunculopontine nucleus of 6OHDA-lesioned rats compared to controls at any survival period. Many of the Fos+ cells within the pedunculopontine and cuneiform nuclei were glutamatergic (35-60%), while none or very few were nitric oxide synthase+. In conclusion, we reveal an increase in the number of strongly labelled Fos+ cells within the cuneiform nucleus of the so-called defensive locomotive system in 6OHDA-lesioned rats. In relation to Parkinson disease, we suggest that this increase is associated with the akinesia or lack of movement seen in patients.

Cell survival patterns in the pedunculopontine tegmental nucleus of methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated monkeys and 6OHDA-lesioned rats: evidence for differences to idiopathic Parkinson disease patients?

We explore the patterns of cell loss in the pedunculopontine tegmental nucleus (PpT), a major locomotor and muscle tone suppression centre of the brainstem, in two animal models of Parkinson disease, namely MPTP (methyl-4-phenyl-1,2,3,6-tetrahydropyridine)-treated monkeys and 6-hydroxydopamine(6OHDA)-lesioned rats. Although there have been many studies documenting the loss of dopaminergic cells from the substantia nigra in these animal models, there has been little, if any, documentation of a loss of cells in the PpT. Results were obtained from macaque monkeys (Macaca fascicularis) and Sprague-Dawley rats. For the monkey series, animals were injected intramuscularly with MPTP (0.2 mg/kg) for 8 days consecutively and then allowed to survive for 21 days thereafter. Each monkey underwent behavioural assessment for parkinsonian symptoms. For the rat series, 6OHDA was injected into the midbrain using stereotactic coordinates. Rats were then allowed to survive for either 7, 14, 28, or 84 days thereafter. Monkey and rat brains were aldehyde-fixed and processed for routine tyrosine hydroxylase (TH; to label nigral dopaminergic cells) and nitric oxide synthase (NOs; to label PpT cholinergic cells) immunocytochemistry. In monkeys, the morphology, distribution and number of NOs(+) cells in the controls and MPTP-treated cases were very similar. In fact, in terms of number, there was only a 1% difference in the mean cell number between the controls and MPTP-treated cases. A comparable pattern was evident in 6OHDA-lesioned rats; there was no substantial difference in morphology, distribution and number of NOs(+) cells on the 6OHDA-lesioned cases when compared to the controls at each of the survival periods post-surgery. In summary, we show no loss of the large cholinergic/NOs(+) cells in the PpT in two animal models of Parkinson disease. This is in contrast to the heavy loss of these cells reported by previous findings in idiopathic Parkinson disease in patients.

Reduction in parvalbumin expression in the zona incerta after 6OHDA lesion in rats.

In an effort to understand better the neurochemical changes that occur in Parkinson disease, we have examined the expression patterns of the calcium-binding protein parvalbumin in the zona incerta in parkinsonian rats. Sprague-Dawley rats had small volumes of either saline (control) or 6 hydroxydopamine (6OHDA) injected into the medial forebrain bundle, the major tract carrying dopaminergic nigrostriatal axons. After various post-lesion survival periods, ranging from 2 hrs to 84 days, rats were perfused with formaldehyde and their brains processed for routine tyrosine hydroxylase (TH) or parvalbumin immunocytochemistry. In the 3 to 84 days post-lesion cases, there was an overall 50% reduction in the number of parvalbumin(+) cells in the zona incerta on the 6OHDA-lesioned side when compared to control. In the 2 hrs post-lesion cases, there was no substantial loss of parvalbumin(+) cells in the zona incerta after 6OHDA lesion, although in these cases (unlike the longer survival periods), there was limited loss of TH(+) cells in the midbrain on the lesion side. The loss of parvalbumin(+) cells from the zona incerta was due to a loss of antigen expression rather than a loss of the cells themselves, since the number of Nissl-stained cells in the zona incerta was similar on the control and 6OHDA-lesioned sides. In summary, our results indicate that a loss of the midbrain dopaminergic cells induces a major change in parvalbumin expression within the zona incerta. This change may have key functional and clinical implications.

Evidence for a glutamatergic projection from the zona incerta to the basal ganglia of rats.

This study explores the organisation and neurochemical nature of the projections from the zona incerta (ZI) to the basal ganglia. Sprague-Dawley rats were anaesthetised with ketamine (100 mg/kg) and Rompun (10 mg/kg), and injections of cholera toxin subunit B were made into each of the following nuclei: the ZI, the substantia nigra (SN), the pedunculopontine tegmental nucleus (PpT), and the entopeduncular nucleus (Ep). Brains were aldehyde fixed, sectioned, and processed using standard methods. Tracer-labelled sections were then doubly labelled with antibodies to glutamate (Glu), nitric oxide synthase (NOS), parvalbumin (Pv), or glutamic acid decarboxylase (GAD; the latter two are markers for GABAergic cells); these neurochemicals characterise most types of ZI cells. After ZI injections, labelling was nonuniform across the different basal ganglia nuclei. The bulk of labelling, both anterograde and retrograde, was seen in the SN and PpT and, to a lesser extent, within the other nuclei of the basal ganglia (e.g., caudate-putamen, globus pallidus, subthalamus, Ep). In the SN, labelling was found in both major parts of the nucleus, the pars compacta and pars reticulata. Within the PpT, however, the bulk of labelling was limited to only one of the two sectors of the nucleus, namely, the pars dissipata (PpTd). The pars compacta of the PpT (PpTc) remained largely free of labelled profiles. After CTb injections into three basal ganglia nuclei (SN, PpT, Ep), most labelled cells in the ZI were glutamate+ and very few were NOS+ or gamma-aminobutyric acidergic. Overall, the results indicate that the ZI is in a position to influence preferentially the activity of the SN and PpTd of the basal ganglia via an excitatory, glutamatergic input.