R. Quirion

Alterations in dendritic morphology of prefrontal cortical and nucleus accumbens neurons in post-pubertal rats after neonatal excitotoxic lesions of the ventral hippocampus

Neonatal ventral hippocampal (nVH) lesions in rats result in adult onset of a number of behavioral and cognitive abnormalities analogous to those seen in schizophrenia, including hyperresponsiveness to stress and psychostimulants and deficits in working memory, sensorimotor gating and social interaction. Molecular and neurochemical alterations in the prefrontal cortex (PFC) and nucleus accumbens (NAcc) of nVH-lesioned animals suggest developmental reorganization of these structures following neonatal lesions. To determine whether nVH lesions lead to neuronal morphological changes, we investigated the effect of nVH lesion on dendritic structure and spine density of pyramidal neurons of the PFC and medium spiny neurons of the NAcc. Bilateral ibotenic acid-induced lesion of the VH was made in Sprague-Dawley pups at postnatal day 7 (P7); and at P70, neuronal morphology was quantified by modified Golgi-Cox staining. The results show that length of basilar dendrites and branching and the density of dendritic spines on layer 3 pyramidal neurons were significantly decreased in rats with nVH lesions. Medium spiny neurons from the NAcc showed a decrease in the density of dendritic spines without significant changes in dendritic length or arborization. The data, comparable to those observed in the PFC of schizophrenic patients, suggest that developmental loss of excitatory projections from the VH may lead to altered neuronal plasticity in the PFC and the NAcc that may contribute to the behavioral changes in these animals.

[3H]1,3-di(2-tolyl)guanidine and [3H](+)pentazocine binding sites in the rat brain: Autoradiographic visualization of the putative sigma1 and sigma2 receptor subtypes

Sigma (sigma) receptors have generated a great deal of interest on the basis of their possible role in psychosis and on locomotor behaviors. The effects of sigma drugs on these various functions are apparently mediated by different sigma receptor subtypes (sigma1 and sigma2). However, little information is currently available on the discrete anatomical distribution of these putative sigma receptor subtypes in the rat brain. The aim of the present study was to investigate, by quantitative autoradiography, the respective distribution of purported sigma1 and sigma2 receptor subtypes in the rat brain using [3H]1,3-di(2-tolyl)guanidine, a universal sigma ligand, and [3H](+)pentazocine, a selective sigma1 ligand. Putative sigma2 receptor sites were visualized using [3H]1,3-di(2-tolyl)guanidine in presence of a saturating concentration of (+)pentazocine. Specific [3H]1,3-di(tolyl)guanidine and [3H](+)pentazocine binding sites were found to be widely but discretely distributed in the rat brain. The highest densities of specific labeling were seen in various cranial nerve nuclei, followed by certain hippocampal sub-fields and laminae, the red nucleus, the interpeduncular nucleus and mid-layers of primary and secondary motor cortices. Lower amounts of specific binding were present in various other structures including most thalamic and hypothalamic nuclei, and the cerebellum. Interestingly, [3H]1,3-di(2-tolyl)guanidine binding in the motor cortex was found to be particularly resistant to a saturating concentration of (+)pentazocine suggesting an enrichment in the putative sigma2 receptor subtype. This also applies for a few other structures such as the nucleus accumbens, substantia nigra pars reticulata, central gray matter, occulomotor nucleus and cerebellum. On the other hand, the sigma1 subtype is more abundant in most other regions with the highest densities seen in the dentate gyrus of the hippocampal formation, facial nucleus, and various thalamic and hypothalamic nuclei. The comparative localization of the sigma1 and sigma2 receptor binding sites probably relates to the differential effects of sigma1 and sigma2 drugs in the rat brain.

An in vivo profile of β-endorphin release in the arcuate nucleus and nucleus accumbens following exposure to stress or alcohol

The aim of the present study was to determine the effects of distinct categories of stressors on beta-endorphin (beta-EP) release in the arcuate nucleus (ArcN) and nucleus accumbens (NAcb) using in vivo microdialysis. Adult male rats were implanted with a cannula aimed at either the NAcb or the ArcN. On the day of testing, a 2 mm microdialysis probe was inserted into the cannula, and artificial cerebrospinal fluid was infused at 2.0 microl/min. After three baseline collections, animals either had a clothespin applied to the base of their tail for 20 min (a physical/tactile stressor), were exposed to fox urine odour for 20 min (a psychological stressor/species-specific threat), or were administered 2.4 g ethanol/kg body weight, 16.5% w/v, i.p. (a chemical/pharmacological stressor) with control animals receiving an equivalent volume of saline. Both tail-pinch and fox odour significantly increased beta-EP release from the ArcN (P<0.05), whilst only tail-pinch enhanced beta-EP release from the NAcb (P<0.01). On the other hand, alcohol stimulated beta-EP release in the NAcb as compared with saline-treated controls (P<0.01), but not in the ArcN. Although the increase in extracellular beta-EP produced by the other stressors was relatively rapid, there was a 90-min delay before alcohol administration caused beta-EP levels to exceed that of saline-injected controls. In conclusion, the fact that physical and fear-inducing psychological stressors stimulate beta-EP release in the ArcN and only physical stressors stimulate beta-EP release in the NAcb, indicates that stressors with different properties are processed differently in the brain. Also, an injection of alcohol caused a delayed increase of beta-EP in the NAcb but not the ArcN, indicating that alcohol may recruit a mechanism that is, at least partially, distinct from stress-related pathways.

Morphine treatment induced calcitonin gene-related peptide and substance P increases in cultured dorsal root ganglion neurons

The mechanism of spinal tolerance to the analgesic effects of opiates is unclear at present. We have reported previously that calcitonin gene-related peptide-like immunoreactivity was significantly increased in primary afferents of the spinal dorsal horn during the development of morphine tolerance, suggesting that changes in the level of pain-related neuropeptides in dorsal root ganglion neurons may be involved [Menard D. P. et al. (1996) J. Neurosci. 16, 2342-2351]. In this study, we investigated if in vitro treatment with morphine can mimic the in vivo findings and induce increases in calcitonin gene-related peptide-like immunostaining in cultured dorsal root ganglion neurons from young (three-month-old) and middle-aged (10-month-old) adult rats. Following a repetitive exposure to morphine sulfate (1, 5, 10 microM) for six days, the number of calcitonin gene-related peptide- and substance P-immunoreactive neurons in cultured dorsal root ganglia from three- and 10-month-old rats was significantly increased. A lower concentration (0.5 microM) of morphine induced these increases only in dorsal root ganglion neurons from middle-aged rats. Morphine treatment was also found to increase the number of calcitonin gene-related peptide-immunoreactive neurons possessing multiple, long branches (i.e. with at least one branch >0.5mm). This apparent increase in the number of calcitonin gene-related peptide- and substance P-immunoreactive neurons observed following morphine treatment was blocked by naloxone, an opiate antagonist, indicating the involvement of genuine opioid receptors. No significant change in the number of neuropeptide Y- or galanin-immunoreactive neurons in cultured dorsal root ganglia was detected following any of these treatments. These data suggest that repeated exposure to morphine rather selectively increases calcitonin gene-related peptide- and substance P-like immunoreactivity in cultured dorsal root ganglion neurons. Moreover, the sensitivity to morphine-induced changes is greater in cultured dorsal root ganglion neurons from 10- compared to three-month-old rats. Hence, cultured dorsal root ganglion neurons can provide a model to investigate the cellular and molecular mechanisms underlying alterations in neuropeptide levels following repeated exposure to opiates and their relevance to the development of opioid tolerance.

Effect of MPTP and l-Deprenyl on Antioxidant Enzymes and Lipid Peroxidation Levels in Mouse Brain

Abstract: Excessive free radical formation or antioxidant enzyme deficiency can result in oxidative stress, a mechanism proposed in the toxicity of MPTP and in the etiology of Parkinsons disease (PD). However, it is unclear if altered antioxidant enzyme activity is sufficient to increase lipid peroxidation in PD. We therefore investigated if MPTP can alter the activity of the antioxidant enzymes, superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH‐PX) and the level of lipid peroxidation. l‐Deprenyl, prior to MPTP administration, is used to inhibit MPP+ formation and its subsequent effect on antioxidant enzymes. MPTP induced a threefold increase in SOD activity in the striatum of C57BL/6 mice. No parallel increase in GSH‐PX or CAT activities was observed, while striatal lipid peroxidation decreased. At the level of the substantia nigra (SN), even though increases in CAT activity and reduction in SOD and GSH‐PX activities were detected, lipid peroxidation was not altered. Interestingly, l‐deprenyl induced similar changes in antioxidant enzymes and lipid peroxidation levels, as did MPTP. Taken together, these results suggest that an alteration in SOD activity, without compensatory increases in CAT or GSH‐PX activities, is not sufficient to induce lipid peroxidation.

Insulin-like growth factor-I and its receptor in the frontal cortex, hippocampus, and cerebellum of normal human and Alzheimer disease brains

Assimilated evidence indicates that the neurotoxic potential of amyloid β (Aβ) peptide and an alteration in the level of growth factor(s) may possibly be involved in the loss of neurons observed in the brain of patients suffering from Alzheimer disease (AD), the prevalent cause of dementia in the elderly. In the present study, using receptor binding assays and immunocytochemistry, we evaluated the pharmacological profile of insulin‐like growth factor‐I (IGF‐I) receptors and the distribution of IGF‐I immunoreactivity in the frontal cortex, hippocampus, and cerebellum of AD and age‐matched control brains. In control brains, [125I]IGF‐I binding was inhibited more potently by IGF‐I than by Des(1‐3)IGF‐I, IGF‐II or insulin. The IC50 values for IGF‐I in the frontal cortex, hippocampus, and cerebellum of the normal brain did not differ significantly from the corresponding regions of the AD brain. Additionally, neither KD nor Bmax values were found to differ in the hippocampus of AD brains from the controls. At the regional levels, [125I]IGF‐I binding sites in the AD brain also remained unaltered compared to the controls. As for the peptide itself, IGF‐I immunoreactivity, in normal control brains, was evident primarily in a subpopulation of astrocytes in the frontal cortex and hippocampus, and in certain Purkinje cells of the cerebellum. In AD brains, a subset of Aβ‐containing neuritic plaques, apart from astrocytes, exhibit IGF‐I immunoreactivity. These results, taken together, suggest a role for IGF‐I in compensatory plasticity and/or survival of the susceptible neurons in AD brains. Synapse 38:450–459, 2000.

ACETYLCHOLINE RELEASE IS ELICITED IN THE VISUAL CORTEX, BUT NOT IN THE PREFRONTAL CORTEX, BY PATTERNED VISUAL STIMULATION: A DUAL IN VIVO MICRODIALYSIS STUDY WITH FUNCTIONAL CORRELATES IN THE RAT BRAIN

By its projections to the primary visual and the prefrontal cortices, the basal forebrain cholinergic system is involved in cognitive processing of sensory stimuli. It has been suggested that visual stimulus-induced cholinergic activation of the visual cortex may exert a permissive role on thalamocortical inputs. However, it is not known if visual stimulation elicits cholinergic activation of high-order brain areas in the absence of attentional need. In the present study, we measured the effects of patterned visual stimulation (horizontal grating) on the release of acetylcholine with dual-probe in vivo microdialysis in the visual and the prefrontal cortices of anesthetized rats. We also used retrograde tracing to determine the anatomical relationships of cholinergic neurons with neurons of the visual system and the prefrontal cortex. Finally, we evaluated a functional correlate of this stimulation, namely c-fos immunolabeling. Patterned visual stimulation elicited significant increases in acetylcholine release in the visual cortex, accompanied by an increased number of c-fos immunoreactive neurons in this brain area. In contrast, in the prefrontal cortex, neither the level of acetylcholine release nor the number of c-fos immunoreactive neurons was significantly changed because of the stimulation. Cholinergic basal forebrain neurons projecting to the visual or the prefrontal cortices were both localized within the horizontal limb of the diagonal band of Broca but were not immunoreactive for c-fos during visual stimulation. No parts of the visual system were found to directly project to these basal forebrain neurons. These results suggest the differential involvement of cholinergic projections in the integration of sensory stimuli, depending on the level of activity of the targeted cortical area.

Quantitative autoradiographic localization of [125I-TYR8]bradykinin receptor binding sites in the rat spinal cord: Effects of neonatal capsaicin, noradrenergic deafferentation, dorsal rhizotomy and peripheral axotomy

In vitro receptor autoradiography was used to localize, quantify and characterize [125I-Tyr8]bradykinin binding sites in all major spinal cord segments of normal rats and animals subjected to various chemical treatments and surgical lesions. [125I-Tyr8]bradykinin specific binding sites were predominantly located to superficial laminae of the rat dorsal horn, with the substantia gelatinosa showing the highest density of labelling (values ranging from 3.1 fmol/mg tissue in cervical to 4.5 fmol/mg tissue in lumbar segments). A moderate density (1.8-3.0 fmol/mg tissue) of specific binding was observed in lamina III, whereas in other areas, i.e. laminae I and IV-X, lower amounts of labelling were detected. Within the superficial laminae of the dorsal horn, [125I-Tyr8]bradykinin binding was largely distributed over the neurophil with some perikarya showing concentrations of labelling. In contrast, the ventral horn showed a rather homogeneous distribution of [125I-Tyr8]bradykinin binding over the neuropil, with silver grain alignments surrounding motoneuron perikaryas and proximal processes. Bradykinin, [Tyr8]bradykinin and B2 receptor antagonists (D-Arg[Hyp3,Thi5,D-Tic7,Oic8]bradykinin (Hoe 140), D-Arg[Tyr3,D-Phe7,Leu8]bradykinin, D-Arg[Hyp3, Leu8]bradykinin, D-Arg[Hyp2, Thi5,8,-Phe7]bradykinin D-Arg[Hyp3, D-Phe7, Leu8]bradykinin, Tyr0, D-Arg[Hyp3, D-Phe7, Leu8]bradykinin inhibited [125I-Tyr8]-bradykinin binding with very high subnanomolar affinities, while the B1 receptor agonist (Tyr0,des-Arg10-kallidin) and antagonist ([Leu8]-des-Arg9-bradykinin) did not significantly affect [125I-Tyr8]bradykinin binding at up to micromolar concentrations. Two weeks after unilateral lumbar dorsal rhizotomy (L1-L6) or peripheral lesions of the sciatic nerve, significant decreases ( +/- 50%) in [125I-Tyr8]bradykinin binding sites were found in ipsilateral laminae I-III of lumbar spinal cord.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of neuropeptide Y receptor subtypes in the normal human brain, including the hypothalamus

The aim of the present study was to investigate the existence and distribution of neuropeptide Y receptor subtypes in various regions of the normal human brain using the peptide YY derivative receptor probes, [125I][Leu31,Pro34]polypeptide YY/Y1 and [125I]polypeptide YY(3-36)/Y2, in addition to the non-selective ligand [125I]polypeptide YY. Membrane binding assays performed with post mortem frontal cortex homogenates revealed that [125I]polypeptide YY and [125I]polypeptide YY(3-36) bound in a time- and protein concentration-dependent manner. Very low amounts of specific [125I][Leu31,Pro34]polypeptide YY binding could be detected even in the presence of high amounts of protein, contrasting with results obtained with [125I]polypeptide YY and [125I]polypeptide YY(3-36), a preferential Y2 receptor probe. Analysis of saturation isotherms revealed that [125I]polypeptide YY(3-36) bound to a single class of high-affinity sites (0.5-2 nM). Significantly higher binding capacities were evident for [125I]polypeptide YY(3-36) as compared to [125I][Leu31,Pro34]polypeptide YY, suggesting that the human frontal cortex, in contrast to the rat, is mostly enriched with Y2 receptors. Ligand selectivity profile confirmed the hypothesis that polypeptide YY(3-36), neuropeptide Y and polypeptide YY but not the [Leu31,Pro34] derivatives are potent competitors of [125I]polypeptide YY and [125I]polypeptide YY(3-36) binding sites. Autoradiographic studies demonstrated further that cortical areas, as well as most other regions of the human brain, are particularly enriched with Y2/[125I]polypeptide YY(3-36) sites, while only low to very low amounts of Y1 binding were detected except in the dentate gyrus of the hippocampal formation. In the human hypothalamus, a preponderance of Y2 binding sites was also noted. Taken together, these results clearly establish that the distribution of the Y1 and Y2 receptor subtypes in human is different from the rodent brain, the Y2 subtype being most abundant in the human brain.

Alteration of expression levels of neuronal nitric oxide synthase and haem oxygenase-2 messenger RNA in the hippocampi and cortices of young adult and aged cognitively unimpaired and impaired Long-Evans rats

Neuronal nitric oxide synthase and haem oxygenase-2 are postulated to be important enzymes involved in neuronal transmission and modulation of free radical levels in neurons. Hippocampal and cortical neuronal nitric oxide synthase and haem oxygenase-2 expressions were compared in young adult (6 months) and aged (24-26 months) Long-Evans rats. Aged rats were assigned as either cognitively unimpaired or impaired based on their performances in the Morris water maze behavioural task. In situ hybridization revealed increased neuronal nitric oxide synthase messenger RNA levels in selected regions of the hippocampi and cortices of aged rats. Moreover, aged cognitively impaired animals showed significantly higher neuronal nitric oxide synthase messenger RNA expression than aged cognitively unimpaired animals in several brain regions. For haem oxygenase-2 mRNA expressions, both young and aged cognitively impaired rats showed increased expressions in hippocampi compared with aged cognitively unimpaired rats, while no difference was found in cortices between all three animal groups. The increase in neuronal nitric oxide synthase messenger RNA expression levels in the aged animals may be related to increased free radical production occurring in ageing. Alternatively, elevated neuronal nitric oxide synthase and haem oxygenase-2 messenger RNA expressions may represent compensatory responses to oxidative stress and age-related changes in neuronal functions. Regarding cognitive status, aged cognitively impaired rats showed significant spatial memory deficits relative to young and aged cognitively unimpaired rats. Our data suggest a correlation between age-related cognitive impairment and change in messenger RNA expressions for the neuronal nitric oxide synthase and haem oxygenase-2 systems in brain areas implicated in learning and memory processes.