Justin D. Oh

Serotonin 5-HT1A agonist improves motor complications in rodent and primate parkinsonian models.

Background: Serotoninergic transmission in the basal ganglia is known to influence dopaminergic mechanisms and motor function. Objective: To evaluate the possibility that serotoninergic 5-HT1A autoreceptors (by regulating the release of serotonin as well as dopamine formed from exogenous levodopa) affect the response alterations complicating levodopa treatment of PD. Methods: The 5-HT1A receptor agonist sarizotan (EMD128130) was systemically administered alone and together with levodopa to parkinsonian rats and nonhuman primates. Results: In 6-hydroxydopamine-lesioned rats, sarizotan (2.5 mg/kg PO) had no effect on the acute rotational response to levodopa but did attenuate the shortening in motor response duration induced by chronic levodopa treatment. In 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-lesioned monkeys, sarizotan (2 mg/kg PO) alone had no effect on parkinsonian severity or on the antiparkinsonian response to levodopa. In contrast, the same dose of sarizotan reduced levodopa-induced choreiform dyskinesias by 91 ± 5.9%. In both species, the motoric effects of sarizotan were blocked by the selective 5-HT1A antagonist WAY100635 (0.1 mg/kg SC), indicating that the observed sarizotan responses were probably mediated at the 5-HT1A autoreceptor. Conclusion: Pharmaceuticals acting to stimulate 5-HT1A receptors could prove useful in the treatment of the motor response complications in parkinsonian patients.

Striatal dopamine- and glutamate-mediated dysregulation in experimental parkinsonism

Characteristic changes involving interactions between dopamine and glutamate in striatal medium spiny neurons now appear to contribute to symptom production in Parkinsons disease (PD). The balance between kinase and phosphatase signaling modifies the phosphorylation state of glutamate receptors and thus their synaptic strength. Sensitization of spiny-neuron NMDA and AMPA receptors alters cortical glutamatergic input to the striatum and modifies striatal GABAergic output, and thus motor function. Conceivably, the pharmacological targeting of spiny-neuron mechanisms modified in PD will provide a safer and more effective therapy.

Enhanced tyrosine phosphorylation of striatal NMDA receptor subunits : Effect of dopaminergic denervation and L-DOPA administration

Sensitization of striatal N-methyl-D-aspartate receptors (NMDAR) has been linked to events leading to the motor response changes associated with the administration of dopaminomimetics to parkinsonian animals and patients. To determine whether tyrosine phosphorylation of NMDAR subunits contributes to the apparent long-term enhancement in synaptic efficacy of these receptors, we examined the effect of unilateral nigrostriatal dopamine system ablation with 6-hydroxydopamine followed by twice-daily treatment with l-DOPA on the phosphorylation state of rat striatal NR2A and NR2B subunits. Three weeks of intermittent l-DOPA administration produced a shortening in the duration of the rotational response to dopaminergic challenge and other changes mimicking those occurring in patients with Parkinsons disease. Concurrently, tyrosine phosphorylation of NR2A and especially of NR2B subunits increased ipsilateral to the lesion (20+/-5% and 46+/-7% of intact striatum, respectively; p<0.01) without attendant changes in subunit protein levels. Selective blockade of NR2B subunits with ACEA 10-1244, but not of NR2A subunits with MDL 100,453, reversed the l-DOPA-induced response alterations. The intrastriatal injection of a tyrosine kinase inhibitor, genistein, at a dose (2.0 microg) that normalized the response shortening, attenuated the NR2A and NR2B phosphorylation increase by about 12% and 24%, respectively (p<0.01). Taken together, these results suggest that augmented tyrosine phosphorylation of NR2B subunits, alone or in combination with the smaller rise in NR2A subunit phosphorylation, contributes to the apparent enhancement in striatal NMDAR sensitivity and thus to the plastic alterations in dopaminergic responses in l-DOPA-treated parkinsonian rats.

Cholinergic neurons in the rat central nervous system demonstrated by in situ hybridization of choline acetyltransferase mRNA

Digoxigenin-labeled RNA probes and in situ hybridization histochemistry were used to examine choline acetyltransferase gene expression in the rat central nervous system. Hybridization signal was present only in brain sections processed with the antisense riboprobe. The sense probe did not yield labeling, further validating the specificity of tissue reactivity. Telencephalic neurons containing the mRNA for the cholinergic synthetic enzyme were found in the caudate-putamen nucleus, nucleus accumbens, olfactory tubercule, islands of Calleja complex, medial septal nucleus, vertical and horizontal limbs of the diagonal band, substantia innominata, nucleus basalis, and nucleus of the ansa lenticularis. Some somata evincing hybridization signal were observed in the anterior amygdalar area, and an occasional such cell was seen in the basolateral and central amygdalar nuclei. Neurons in the cerebral cortex, hippocampus, and primary olfactory structures did not demonstrate hybridocytochemically detectable amounts of choline acetyltransferase mRNA. Thalamic cells were devoid of reactivity, with the exception of several neurons located primarily in the ventral two-thirds of the medial habenula. A few somata labeled with riboprobe were found in the lateral hypothalamus, caudal extension of the internal capsule, and zona incerta. Neurons in the pedunculopontine and laterodorsal tegmental nuclei were moderately reactive, whereas cells of the parabigeminal nucleus exhibited a very weak hybridization signal. No somata in the brainstem raphe nuclei, including raphe obscurus and raphe magnus, were observed to bind riboprobe. In contrast, motor neurons of the cranial nerve nuclei demonstrated relatively large amounts of choline acetyltransferase mRNA. Putative cholinergic somata in the ventral horns and intermediolateral cell columns of the spinal cord were also labeled with riboprobe, as were a few cells around the central canal. We conclude that hybridocytochemistry with digoxigenin-labeled riboprobes confirms the existence of cholinergic neurons (i.e. those that synthesize and use acetylcholine as a neurotransmitter) in most of the neural regions deduced to contain them on the basis of previous histochemical and immunocytochemical data. Notable exceptions are the cerebral cortex and hippocampus, which do not possess neurons expressing detectable levels of choline acetyltransferase mRNA.

Effect of dopamine denervation and dopamine agonist administration on serine phosphorylation of striatal NMDA receptor subunits.

Sensitization of striatal N-methyl-d-aspartate (NMDA) receptors has been implicated in the pathogenesis of the response alterations associated with dopaminomimetic treatment of parkinsonian animals and patients. To determine whether serine phosphorylation of NMDA receptor subunits by activation of Ca2+/calmodulin-dependent protein-kinase II (CaMKII) contributes to this process, we examined the effects of unilateral nigrostriatal ablation with 6-hydroxydopamine and subsequent treatment with levodopa, SKF 38393 (D1-preferring dopamine agonist), or quinpirole (D2-preferring agonist) on motor responses and phosphorylation states. Three weeks of twice-daily levodopa administration to rats shortened the duration of their rotational response to levodopa or SKF 38393 challenge, but prolonged the duration of quinpirole-induced rotation. At the same time, levodopa treatment elevated serine phosphorylation of striatal NR2A (p<0.02), but not that of NR2B subunits, without associated changes in subunit protein levels. Chronic treatment with SKF 38393 increased NR2A (p<0.0001) but decreased NR2B (p<0.004) serine phosphorylation. In contrast, chronic quinpirole treatment had no effect on NR2A but increased NR2B phosphorylation (p<0.0001). The acute intrastriatal injection of the CaMKII inhibitor KN93 (1.0 micrograms) not only normalized the levodopa-induced motor response alterations but also attenuated the D1 and D2 receptor-mediated serine phosphorylation of NR2A and NR2B subunits, respectively (p<0.02). These results suggest that a CaMKII-mediated rise in serine phosphorylation of NMDA receptor subunits induced by intermittent stimulation of D1 or D2 dopaminergic receptors contributes to the apparent enhancement in striatal NMDA receptor sensitivity and thus to the dopaminergic response plasticity in levodopa-treated parkinsonian rats.

Antiparkinsonian and antidyskinetic activity of drugs targeting central glutamatergic mechanisms

Abstract Motor dysfunction produced by the chronic non-physiological stimulation of dopaminergic receptors on striatal medium spiny neurons is associated with alterations in the sensitivity of glutamatergic receptors, including those of the N-methyl-D-aspartate (NMDA) subtype. Functional characteristics of these ionotropic receptors are regulated by their phosphorylation state. Lesioning the nigrostriatal dopamine system of rats induces parkinsonian signs and increases the phosphorylation of striatal NMDA receptor subunits on serine and tyrosine residues. The intrastriatal administration of certain inhibitors of the kinases capable of phosphorylating NMDA receptors produces a dopaminomimetic motor response in these animals. Treating parkinsonian rats twice daily with levodopa induces many of the characteristic features of the human motor complication syndrome and further increases the serine and tyrosine phosphorylation of specific NMDA receptor subunits. Again, the intrastriatal administration of selective inhibitors of certain serine and tyrosine kinases alleviates the motor complications. NMDA receptor antagonists, including some non-competitive channel blockers, act both palliatively and prophylactically in rodent and primate models to reverse these levodopa-induced response alterations. Similarly, in clinical studies dextrorphan, dextromethorphan, and amantadine have been found to be efficacious against motor complications. Recent observations in animal models further indicate that certain amino-3-hydroxy-5-methyl-4-isoxazole proprionic acid (AMPA) antagonists alleviate, while others exacerbate, these complications. Thus, it appears that the denervation or intermittent stimulation of striatal dopaminergic receptors differentially activates signal transduction pathways in medium spiny neurons. These in turn modify the phosphorylation state of ionotropic glutamate receptors and consequently their sensitivity to cortical input. These striatal changes contribute to symptom production in Parkinson’s disease, and their prevention or reversal could prove useful in the treatment of this disorder.

Organization of central cholinergic neurons revealed by combined in situ hybridization histochemistry and choline-O-acetyltransferase immunocytochemistry

Digoxigenin-labeled riboprobes and in situ hybridization of choline-O-acetyltransferase mRNA, both alone and in combination with immunohistochemical procedures for the synthetic enzyme of acetylcholine, were used to map the topography of putative cholinergic neurons in the rat central nervous system. Only the anti-sense riboprobe yielded specific labeling, which was absent in brain sections processed with sense riboprobe. Telencephalic neurons demonstrating the mRNA for choline-O-acetyltransferase and choline-O-acetyltransferase-like immunoreactivity were found in the caudate-putamen nucleus, nucleus accumbens, olfactory tubercule, Islands of Calleja complex, medial septal nucleus, vertical and horizontal limbs of the diagonal band, substantia innominata, nucleus basalis, and nucleus of the ansa lenticularis, as well as occasionally in the amygdala. Neurons in the cerebral cortex, hippocampus, and primary olfactory structures did not demonstrate hybridization signal, even though some cells in those areas were observed to exhibit choline-O-acetyltransferase-like immunopositivity. Thalamic cells were devoid of hybrido- and immunoreactivity, with the exception of several neurons located primarily in the ventral two-thirds of the medial habenula. A few cell bodies labeled with riboprobe and co-localizing choline-O-acetyltransferase-like immunopositivity were found in the lateral hypothalamus, caudal extension of the internal capsule, and zona incerta. Neurons in the pedunculopontine and laterodorsal tegmental nuclei evinced moderate hybridization signal, whereas cells of the parabigeminal nucleus were very weakly reactive. In contrast, motor neurons of the cranial nerve nuclei demonstrated high levels of choline-O-acetyltransferase mRNA and choline-O-acetyltransferase-like immunoreactivity. Putative cholinergic somata in the ventral horns and intermediolateral cell columns of the spinal cord and around the central canal were also labeled with riboprobe. It is concluded that hybridocytochemistry with digoxigenin-labeled riboprobes confirms the existence of cholinergic neurons in most of the neural regions believed to contain them on the basis of acetylcholinesterase pharmacohistochemistry and choline-O-acetyltransferase immunocytochemistry, with the prominent exceptions of the cerebral cortex, hippocampus, olfactory bulb, anterior olfactory nucleus, and caudal raphe nuclei, which apparently do not possess neurons expressing detectable levels of the mRNA for the synthetic enzyme of acetylcholine.

Overexpression of neurotrophin receptor p75 contributes to the excitotoxin-induced cholinergic neuronal death in rat basal forebrain.

Both excitotoxicity and altered trophic factor support have been implicated in the pathogenesis of Alzheimers disease. To determine whether stimulation of p75, the low-affinity receptor for nerve growth factor, contributes to the excitotoxin-induced apoptotic death of cholinergic neurons, we examined the effect of unilateral kainic acid (KA; PBS vehicle, 1.25, 2.5 and 5.0 nmol) administration into rat basal forebrain on neuronal loss and p75 expression. KA (2. 5 nmol) destroyed 43% of Nissl-stained neurons and 70% of choline acetyltransferase (ChAT)-positive neurons 5 days after injection. Agarose gel electrophoresis revealed that KA (2.5 nmol) induced local internucleosomal DNA fragmentation after 6-48 h. Immunohistochemical analysis further showed that KA (2.5 nmol) augmented p75 immunoreactivity at a time when terminal transferase-mediated deoxyuridine trophosphate (d-UTP)-digoxigenin nick end labeling (TUNEL)-positive nuclei were increased. Many fragmented nuclei were co-labeled with ChAT antibody. The chronic administration of anti-rat p75 or the protein synthesis inhibitor, cycloheximide, but not anti-human p75, substantially reduced the KA-induced destruction of cholinergic neurons and the induction of internucleosomal DNA fragmentation. Anti-rat p75, but not cycloheximide, also reversed the spatial memory impairment produced by KA. These findings suggest that overexpression of p75 contributes to the excitotoxin-induced death of rat basal forebrain cholinergic neurons by an apoptotic-like mechanism.

Choline Acetyltransferase mRNA Plasticity with Pavlovian Conditioning

Choline acetyltransferase mRNA and somal area increased selectively in the ventral nucleus basalis of rats trained that a tone signals immediate shock (i.e., predicts danger). Retrograde tracing showed the affected cells correspond to those that project to the auditory cortex. Behavior responses and mRNA increased significantly above those of control groups trained with the tone not signaling immediate shock. In one of those control groups, animals learned that the same tone signaled a shock-free period before shock. These animals showed a visibly decreased riboprobe and a trend toward smaller somal areas. These results implicate transcriptional regulation of choline acetyltransferase in long-term memory storage. Selective attention and inattention to the tone are possible components of memory encoded by the molecular changes reported here.

Intrafimbrial colchicine produces transient impairment of radial-arm maze performance correlated with morphologic abnormalities of septohippocampal neurons expressing cholinergic markers and nerve growth factor receptor.

The microtubule disrupting agent, colchicine, was infused both bilaterally and unilaterally into the fimbria of the rat brain. Such infusions produced a transient impairment in radial-arm maze performance during the first week following surgery but only in bilaterally injected animals. This behavioral finding was correlated with a reduction in the number of neurons expressing choline acetyltransferase and nerve growth factor receptor in the septum and vertical limb of the diagonal band but not in other regions of the basal nuclear complex. The altered expression of the two neurochemical markers was not due to cellular degeneration because numbers of neurons demonstrated by Nissl staining were unchanged. Putative cholinergic fibers in the fimbria demonstrating acetylcholinesterase and nerve growth factor receptor also showed aberrations in caliber, shape, and course.