Maria Morello

Colocalization of somatostatin, neuropeptide Y, neuronal nitric oxide synthase and NADPH-diaphorase in striatal interneurons in rats

The neuropeptides somatostatin (SS), neuropeptide Y (NPY), the enzyme neuronal nitric oxide synthase (nNOS) and enzymatic activity for NADPH diaphorase (NADPHd) are extensively colocalized in striatal interneurons, which has led to the widespread tendency to operationally treat all four substances as being completely colocalized within a single class of striatal interneurons. We have explored the validity of this assumption in rat striatum using multiple-labeling methods. Conventional epi-illumination fluorescence microscopy was used to examine tissue triple labeled for SS, NPY and nNOS, or double-labeled for SS and nNOS or for SS and NPY. In tissue double-labeled for SS and nNOs, confocal laser scanning microscopy (CLSM) images of SS and nNOS labeling were compared to subsequent NADPHd labeling. We found that SS, NPY and nNOS co-occurred extensively, but a moderately abundant population of neurons containing SS and nNOS but not NPY was also observed, as were small populations of SS only and nNOS only neurons. About 80% of SS+ neurons contained NPY, and no NPY neurons were devoid of SS or nNOS. All neurons containing nNOS in rat striatum were found to contain NADPHd. Combining our various quantitative observations, we found that of those striatal neurons containing any combination of SS, NPY, nNOS and NADPHd in rats, about 73% contained all four, 16% contained SS, nNOS and NADPHd, 5% contained SS only, and 6% contained only nNOS and NADPHd. These results indicate that while there is a large population of striatal neurons in which SS, NPY, nNOS and NADPHd are colocalized in rats, there may be smaller populations of striatal neurons devoid of NPY in which SS or nNOS/NADPHd are found individually or together.

A Synaptic Mechanism Underlying the Behavioral Abnormalities Induced by Manganese Intoxication

In the present study we have characterized a rat model of manganese (Mn) intoxication leading to behavioral disinhibition in the absence of major motor alterations. These behavioral changes were associated with significantly increased brain Mn levels but were uncoupled to anatomical lesions of the striatum or to morphological and cytochemical changes of the nigrostriatal dopaminergic pathway. The analysis of this model at cellular level showed an enhanced dopaminergic inhibitory control of the corticostriatal excitatory transmission via presynaptic D2-like dopamine (DA) receptors in slices obtained from Mn-treated rats. Conversely, the use of agonists acting on presynaptic purinergic, muscarinic, and glutamatergic metabotropic receptors revealed a normal sensitivity. Moreover, membrane responses recorded from single dopaminergic neurons following activation of D2 DA autoreceptors were also unchanged following Mn intoxication. Thus, our findings indicate a selective involvement of the D2-like DA receptors located on glutamatergic corticostriatal terminals in this pathological condition and suggest that the behavioral symptoms described in the early clinical phase of manganism may be caused by an abnormal dopaminergic inhibitory control on corticostriatal inputs. The identification of the synaptic mechanism underlying the early phase of Mn intoxication might have a critical importance to understand the causes of the progression of this pathological condition towards an established phase characterized by motor abnormalities and anatomical lesions of the basal ganglia.

Down-regulation of nitrergic transmission in the rat striatum after chronic nigrostriatal deafferentation.

Dopamine and NO are physiological stimulators of synthesis of cAMP and cGMP, respectively, and NOu2003synthase‐containing interneurons in the striatum are physiologically activated by dopamine‐containing neurons in the substantia nigra. This study investigated whether lesioning dopamine neurons has multiple consequences in the striatum consistent with the reported sensitization of cAMP synthesis, including alteration of the NO–cGMP pathway and phosphodiesterase‐dependent metabolism of cyclic nucleotides. The substantia nigra of adult Sprague‐Dawley rats was unilaterally lesioned with 6‐hydroxydopamine. Two months later, we determined expression of NOu2003synthase and evaluated cGMP and cAMP levels of intact and deafferented striatum. Moreover, we evaluated cAMP– and cGMP–phosphodiesterase activities in basal conditions and after Ca2+–calmodulin stimulation and determined the expression of the phosphodiesterase‐1B isoform and the levels of phosphodiesterase‐1B mRNA. Using immunocytochemistry we characterized the distribution of NOu2003synthase and phosphodiesterase‐1B within striatal neurons. In the dopamine‐deafferented striatum, NOu2003synthase levels were decreased by 42% while NOu2003synthase‐immunopositive intrastriatal fibres but not NOu2003synthase neuronal bodies were reduced in number. In the deafferented striatum basal cGMP levels were reduced, and cAMP levels were increased, but cGMP–phosphodiesterase and cAMP–phosphodiesterase activities were both increased in basal and Ca2+–calmodulin‐stimulated conditions. Accordingly, phosphodiesterase‐1B expression and phosphodiesterase‐1B mRNA were upregulated while a large population of medium‐sized striatal neurons showed increased phosphodiesterase‐1B immunoreactivity. Dopamine deafferentation led to a complex down‐regulation of the NO–cGMP pathway in the striatum and to an up‐regulation of phosphodiesterase‐1B‐dependent cyclic nucleotide metabolism, showing new aspects of neuronal plasticity in experimental hemiparkinsonism.

Selective vulnerability of pallidal neurons in the early phases of manganese intoxication

Abstract. Prolonged exposure to manganese in mammals may cause an extrapyramidal disorder characterized by dystonia and rigidity. Gliosis in the pallidal segments underlies the well-established phase of the intoxication. The early phase of the intoxication may be characterized by psychic, nonmotor signs, and its morphological and electrophysiological correlates are less defined. In a rat model of manganese intoxication (20xa0mg/ml in drinking water for 3xa0months), neither neuronal loss nor gliosis was detected in globus pallidus (GP). However, a striking vulnerability of manganese-treated GP neurons emerged. The majority of GP neurons isolated from manganese-treated rats died following brief incubation in standard dissociation media. In addition, patch-clamp recordings in the whole-cell configuration were not tolerated by surviving GP neurons. Neither coeval but untreated GP neurons nor striatal ones manifested analogous susceptibility. Using the perforated-patch mode of recording we attempted at identifying the functional hallmarks of GP vulnerability: in particular, voltage-gated calcium currents and glutamate-induced currents were examined. Manganese-treated GP neurons exhibited calcium currents similar to control cells aside from a slight reduction in the dihydropyridine-sensitive current facilitation. Strikingly, manganese-treated GP cells – but not striatal ones – manifested peculiar responses to glutamate, since repeated applications of the excitatory amino acid, at concentrations which commonly promote desensitizing responses, produced instead an irreversible cell damage. Possible mechanisms are discussed.

Nitric oxide inhibition aggravates ischemic damage of hippocampal but not of NADPH neurons in gerbils.

BACKGROUND AND PURPOSEnNitric oxide may influence pathophysiology of brain ischemia in a complex way depending on the sources of its production either from neurons or endothelial cells. We investigated whether inhibition of nitric oxide synthesis affects postischemic neuronal death in hippocampus. Moreover, we evaluated whether the presence of nitric oxide synthase activity in specific neurons protects these against ischemia in the hippocampus, striatum, and sensorimotor cortex.nnnMETHODSnTo inhibit nitric oxide synthase, several dosing regimens of NG-nitro-L-arginine methyl ester (L-NAME) were used (5 or 50 mg/kg IP, twice a day for 4 days, or 30 mg/kg IV) in gerbils. Control animals received either the isomer NG-nitro-D-arginine methyl ester or the vehicle. The gerbils underwent 10-minute occlusion of carotid arteries under ether anesthesia and controlled body temperature while physiological parameters were monitored. Neuronal damage was assessed 5 days after ischemia using Nissl-stained sections of hippocampus. Nitric oxide synthase neurons were histochemically stained for reduced nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase activity.nnnRESULTSnL-NAME treatments, but not the chronic one at 5 mg/kg, induced elevation of blood pressure (30% to 80% greater than the control level, P < .01), as observed shortly before and after bilateral carotid occlusion. Postischemic neuronal loss in the CA1 through CA4 sectors was worsened by chronic pretreatment with L-NAME at 50 mg/kg (eg, CA1 neuronal counts per 100-microns length: 3.2 +/- 2.74, mean +/- SD; n = 19; P < .01). After the acute (30 mg/kg) or chronic pretreatment at lower dosage (5 mg/kg) with L-NAME, neuronal loss was comparable to that of animals treated with the D-isomer or the vehicle (CA1 counts in vehicle-treated animals: 7.65 +/- 6.51, mean +/- SD; n = 14). None of the L-NAME treatments affected postischemic survival of NADPH diaphorase-positive neurons in hippocampus, striatum, and sensorimotor cortex.nnnCONCLUSIONSnThese observations demonstrate that inhibition of endothelial and neuronal nitric oxide synthase activity does not modify resistance of nitric oxide-producing neurons to transient ischemia. The severe inhibition of nitric oxide production aggravates postischemic neuronal death in the hippocampus, whereas the mild inhibition is ineffective.

Nitrergic neurons make synapses on dual-input dendritic spines of neurons in the cerebral cortex and the striatum of the rat: Implication for a postsynaptic action of nitric oxide

Pre-embedding electron microscopic immunocytochemistry was used to examine the ultrastructure of neurons containing nitric oxide synthase and to evaluate their synaptic relationships with target neurons in the striatum and sensorimotor cerebral cortex. Intense nitric oxide synthase immunoreactivity was found by light and electron microscopy in a type of aspiny neuron scattered in these two regions. The intensity of the labeling was uniform in the soma, dendrites and axon terminals of these neurons. In both forebrain regions, nitric oxide synthase-immunoreactive neurons received synaptic contacts from unlabeled terminals, which were mostly apposed to small-caliber dendrites. The unlabeled symmetric contacts were generally about four times as abundant as the unlabeled asymmetric contacts on the nitric oxide synthase-immunoreactive neurons. Terminals labeled for nitric oxide synthase were filled with synaptic vesicles and were observed to contact unlabeled neurons. Only 54% (in the cerebral cortex) and 44.3% (in the striatum) of the nitric oxide synthase-immunoreactive terminals making apposition with the target structures were observed to form synaptic membrane specializations within the plane of the randomly sampled sections. The most common targets of nitric oxide synthase-immunoreactive terminals were thin dendritic shafts (54% of the immunoreactive terminals in the cortex and 75.7% of the immunoreactive terminals in the striatum), while dendritic spines were a common secondary target (42% of the immunoreactive terminals in the cortex and 20.6% of the immunoreactive terminals in the striatum). The spines contacted by nitric oxide synthase-immunoreactive terminals typically also received an asymmetric synaptic contact from an unlabeled axon terminal. These findings suggest that: (i) nitric oxide synthase-immunoreactive neurons in the cortex and striatum preponderantly receive inhibitory input; (ii) nitric oxide synthase-containing terminals commonly make synaptic contact with target structures in the cortex and striatum; (iii) spines targeted by nitric oxide synthase-containing terminals in the cortex and striatum commonly receive an asymmetric contact as well, which may provide a basis for a synaptic interaction of nitric oxide with excitatory input to individual spines.

Ultrastructural study of nitric oxide synthase-containing striatal neurons and their relationship with parvalbumin-containing neurons in rats

Single- and double-label electron microscopic immunocytochemistry was used to examine the ultrastructure of striatal neurons containing nitric oxide synthase (NOS+) and evaluate the synaptic relationship of NOS+ striatal neurons with those containing parvalbumin (PV+). In both the single-label and double-label studies, NOS+ perikarya were observed to possess polylobulated nuclei. In the single-label studies, NOS+ terminals were seen forming synaptic contacts with dendritic shafts and dendritic spines that did not contain NOS, but not with NOS+ perikarya or dendrites. In the double-label studies (using diaminobenzidine and silver intensified immunogold as markers), nitric oxide synthase and parvalbumin immunoreactions were found in two different populations of medium-sized aspiny striatal neurons. The PV+ axon terminals were seen forming symmetric synapses on the dendritic spines of neurons devoid of PV or NOS labeling, on PV+ dendrites, and on NOS+ soma and dendrites. In contrast, NOS+ terminals were not observed to form synaptic contacts with the dendrites or soma of either PV+ or NOS+ neurons. These findings suggest that NOS+ striatal interneurons form synaptic contact with the spines and presumably the dendrites of striatal projection neurons, but not with the dendrites or soma of PV+ or NOS+ striatal interneurons. NOS+ neurons do, however, receive synaptic input from PV+ neurons.

Striatal modulation of cAMP-response-element-binding protein (CREB) after excitotoxic lesions: Implications with neuronal vulnerability in Huntington's disease

Recent evidence has shown that the activity of cAMP responsive element‐binding protein (CREB) and of CREB‐binding protein (CBP) is decreased in Huntingtons disease (HD) [ Steffan et al. (2000)Proc. Natl Acad. Sci. USA, 97, 6763–6768; Gines et al. (2003)Hum. Mol. Genet., 12, 497–508; Rouaux et al. (2004) Biochem. Pharmacol., 68, 1157–1164; Sugars et al. (2004)J. Biol. Chem., 279, 4988–4999]. Such decrease is thought to reflect the impaired energy metabolism observed in a HD mouse model, where a decline in striatum cAMP levels has been observed [ Gines et al. (2003)Hum. Mol. Genet., 12, 497–508]. Increased levels of CREB have also been demonstrated to exert neuroprotective functions [ Lonze & Ginty (2002)Neuron, 35, 605–623; Lonze et al. (2002)Neuron, 34, 371–385]. Our study aimed to investigate the distribution of CREB in the neuronal subpopulations of the striatum in normal rats compared to the HD model of quinolinic acid lesion. Twenty‐five Wistar rats were administered quinolinic acid 100u2003mm into the right striatum, and killed after 24u2003h, 48u2003h, 1u2003week, 2u2003weeks, and six weeks, respectively. The contralateral striata were used as controls. Dual‐label immunofluorescence was employed using antibodies against phosphorylated CREB and each of the different neuronal subpopulations markers. Our results show that activated CREB levels decrease progressively in projection neurons and parvalbumin (PARV) and calretinin (CALR) interneurons, whereas such levels remain stable in cholinergic and somatostatin interneurons. Thus, we speculate that the ability of cholinergic interneurons to maintain their levels of CREB after excitotoxic lesions is one of the factors determining their protection in Huntingtons disease.

Vitamin D, Cognition and Alzheimer’s Disease: The Therapeutic Benefit is in the D-Tails

Since its discovery during the epidemic of rickets in the early 1920s, the physiological effects of vitamin D on calcium/phosphorus homeostasis have been thoroughly studied. Along with the understanding of its actions on skeletal diseases and advances in cellular and molecular biology, this misnamed vitamin has gained attention as a potential player in a growing number of physiological processes and a variety of diseases. During the last 25 years, vitamin D has emerged as a serious candidate in nervous system development and function and a therapeutic tool in a number of neurological pathologies. More recently, experimental and pre-clinical data suggest a link between vitamin D status and cognitive function. Human studies strongly support a correlation between low levels of circulating 25-hydroxyvitamin D (25(OH)D) and cognitive impairment or dementia in aging populations. In parallel, animal studies show that supplementation with vitamin D is protective against biological processes associated with Alzheimer’s disease (AD) and enhances learning and memory performance in various animal models of aging and AD. These experimental observations support multiple mechanisms by which vitamin D can act against neurodegenerative processes. However, clinical interventional studies are disappointing and fail to associate increased 25(OH)D levels with improved cognitive outcomes. This review collects the current available data from both animal and human studies and discusses the considerations that future studies examining the effects of vitamin D status on neurocognitive function might consider.

A Case of PANDAS Treated With Tetrabenazine and Tonsillectomy

PANDAS (pediatric autoimmune neuropsychiatric disorders associated with streptococcal infections) is a rare clinical syndrome characterized by the presence of tics, Tourette syndrome, obsessive-compulsive disorder, or chorea in the context of an immediately precedent streptococcal infection. In this report, we describe the case of an 11-year-old boy who developed PANDAS with severe choreic movements. The criteria for PANDAS diagnosis were met. Moreover, serum antibrain antibodies were present. The patient was initially treated with tetrabenazine 12.5 mg twice daily with remission of the neurological symptoms. Subsequently, the patient underwent tonsillectomy and has been asymptomatic since, with antistreptolysin O titer levels in range.