Women are more likely to develop Post Stroke Depression (PSD) than men and generally do not respond well to anti-depressants with age. This study investigated the effect of microRNA mir363-3p treatment on PSD using a physiologically-relevant animal model. Our previous work showed that mir363-3p treatment, delivered post-stroke, effectively reduces infarct volume in the acute phase of stroke in middle-aged females but not males. Middle-aged female Sprague Dawley rats were tested for baseline sensory motor function and depressive-like behaviors, and then subjected to ischemic stroke via middle cerebral artery occlusion (MCAo) or sham surgery. Animals received either control oligos (MCAo+scrambled, Sham+scrambled) or mir363-3p (MCAo+mir363-3p, Sham+mir363-3p) treatment 4 h later. Sensory motor function and depressive-like behaviors were reassessed up to 100 d after stroke, and circulating levels of IL-6, TNF-alpha and Brain-Derived Neurotrophic Factor (BDNF) were quantified at regular intervals. Prior to termination, Fluorogold was injected into the striatum to assess meso-striatal projections. MCAo+scrambled animals had impaired sensorimotor performance in the acute phase (5 days) of stroke and developed anhedonia, decreased sociability and increased helplessness in the chronic phase. MCAo+mir363-3p animals showed significantly less sensory motor impairment and fewer depressive-like behaviors. IL-6 and TNF-alpha were elevated transiently at 4 weeks after MCAo in both groups. BDNF levels decreased progressively after stroke in the MCAo+scrambled group, and this was attenuated in the mir363-3p group. The number of retrogradely-labeled SNc and VTA cells was reduced in the ischemic hemisphere of the MCAo+scrambled group. In contrast, there was no interhemispheric difference in the number of retrogradely-labeled SNc and VTA cells of MCAo+mir363-3p treated animals. Our results support a therapeutic role for mir363-3p for long-term stroke disability.

Parkinson's disease (PD) is a common neurodegenerative disorder. To date, the diagnosis of PD relies mainly on clinical manifestations whereas neuropathological confirmation of the brain is only possible with postmortem studies. Neuronal loss in the substantia nigra pars compacta (SNc) associated with Lewy bodies/neurites is the pathological hallmark feature of PD. The major component of Lewy pathology (LP) is misfolded alpha-synuclein (α-SYN). There is evidence that the distribution of LP is not only limited to the brain but extends to peripheral tissues, including gastrointestinal tract, salivary glands, olfactory mucosa, skin, retina, adrenal gland, and heart. Sensitivity and specificity of α-SYN detection in PD vary greatly among studies due to methodological heterogeneity, such as sampling sites and size, tissue preparation, staining techniques, and antibodies used. Of note, α-SYN has also been found in preclinical and prodromal PD. Further in vivo studies focusing on favorable biopsy sites and standard techniques are needed to get better understanding of α-SYN deposits in preclinical, prodromal, and clinical PD.

On the basis of the structure-activity relationships of delta-opioid-selective peptide ligands and on a model of the proposed bioactive conformation for a potent and selective, conformationally constrained delta-opioid peptide ligand [(2S, 3R)-TMT1]DPDPE, a series of small organic peptide mimetic compounds targeted for the delta-opioid receptor have been designed, synthesized, and evaluated in radiolabeled ligand binding assays and in vitro bioassays. The new non-peptide ligands use piperazine as a template to present the most important pharmacophore groups, including phenol and phenyl groups and a hydrophobic moiety. This hydrophobic group was designed to mimic the hydrophobic character of the D-Pen residues in DPDPE, which has been found to be extremely important for increasing the binding affinity and selectivity of these non-peptide ligands for the delta-opioid receptor over the mu-opioid receptor. Compound 6f (SL-3111) showed 8 nM binding affinity and over 2000-fold selectivity for the delta-opioid receptor over the mu-opioid receptor. Both enantiomers of SL-3111 were separated, and the (-)-isomer was shown to be the compound with the highest affinity for the delta-opioid receptor found in our study (IC50 = 4.1 nM), with a selectivity very similar to that observed for the racemic compound. The phenol hydroxyl group of SL-3111 turned out to be essential to maintain high affinity for the delta-opioid receptor, which also was observed in the case of the delta-opioid-selective peptide ligand DPDPE. Binding studies of SL-3111 and [p-ClPhe4]DPDPE on the cloned wild-type and mutated human delta-opioid receptors suggested that the new non-peptide ligand has a binding profile similar to that of DPDPE but different from that of (+)-4-[((alphaR)-alpha(2S,5R)-4-allyl-2, 5-dimethyl-1-piperazinyl)-3-methoxybenzyl]-N,N-diethylbenzamide (SNC-80), another delta-opioid-selective non-peptide ligand.

Kynurenic acid (KYNA), an endogenous antagonist of ionotropic excitatory amino acid (EAA) receptors, was tested for its ability to modulate N-methyl-D-aspartate (NMDA)- and alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)-induced excitation of dopamine (DA)-containing neurons in the zona compacta of the rat substantia nigra (SNc). Experiments were conducted using extracellular recording techniques in conjunction with an in vitro brain slice preparation. Bath application of NMDA (1-20 microM) or AMPA (0.5-10 microM) produced a concentration-dependent increase in the firing rate of SNc DA neurons but had no effect on firing pattern. The highest concentration of both agonists produced a rapid and reversible cessation of activity that was attributed to acute induction of depolarization block. Addition of glycine (GLY) (up to 100 microM) to the bathing solution had no effect on either basal firing rate or the increase in activity produced by NMDA. KYNA (10 microM-1 mM) antagonized the excitatory effects of both NMDA (15 microM) and AMPA (3 microM) in a concentration-dependent fashion (IC50:102 microM and 64 microM, respectively) without affecting basal firing rate. Perfusion of tissue slices with a modified Ringer's solution containing low Mg2+ (0.12 mM) increased NMDA-induced excitation but did not affect the antagonist properties of KYNA. D-serine (100 microM) reversed the ability of KYNA to block the excitatory effects of NMDA, suggesting that KYNA attenuates NMDA-induced excitation of SNc DA neurons via blockade of the GLY allosteric site on the NMDA receptor. The ability of KYNA to modulate the excitatory effects of both NMDA and non-NMDA agonists implies that endogenous KYNA may play a physiological role in regulating DA cell excitability.

Electrophysiological techniques were used to study the effects of the new compound, DAU 6215 ((3-alpha-tropanyl) 1H-benzimidazolone-3-carboxamide chloride), a selective 5-HT3 receptor antagonist, on the activity of dopamine (DA)-containing neurons in the substantia nigra pars compacta (SNc) and ventral tegmental area (VTA). Acute i.v. injections of DAU 6215 did not cause any change in the basal firing rate of DA neurons in the SNc or in the VTA. Pretreatment with DAU 6215 did not modify the inhibitory effect of apomorphine on the firing rate of midbrain DA neurons. Acute s.c. administration of DAU 6215 caused a significant increase in the number of spontaneously active DA neurons in the VTA but not in the SNc. This effect was similar to that of acute clozapine, whereas acute haloperidol caused a significant increase of spontaneously active DA neurons in both the SNc and the VTA. Repeated consecutive s.c. administration of DAU 6215 and clozapine for 21 days produced a significant decrease in the number of spontaneously active DA neurons in the VTA but not in the SNc. Chronic haloperidol (21 days) decreased the number of DA cells both in the SNc and VTA. The effect of chronic DAU 6215 on the activity of VTA DA neurons was reversed by apomorphine, suggesting that these neurons were probably under a state of depolarization block. These findings indicate that DAU 6215 may have potential antipsychotic activity, probably associated with a low incidence of extrapyramidal side-effects.

It has been widely accepted that repeated administration of kappa-opioid receptor agonists leads to the development of antinociceptive tolerance. The present study was designed to investigate the effect of repeated administration of a selective kappa-opioid receptor agonist (1S-trans)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)cyclohexyl]-benzeneacetamide hydrochloride ((-)U-50,488H) on the mu- and delta-opioid receptor agonist-induced antinociception and G-protein activation in mice. The mice were injected either subcutaneously (s.c.) or intracerebroventricularly (i.c.v.) pretreated with saline or (-)U-50,488H once a day for seven consecutive days. Two hours after the last injection, the mice were challenged by either mu- or delta-opioid receptor agonist for the antinociceptive assay. Repeated treatment with (-)U-50,488H (s.c. or i.c.v.) significantly enhanced antinociceptive effect of both mu-opioid receptor agonist (morphine) and delta-opioid receptor agonists ([d-Ala2]deltorphin (DELT) and (+)-4-[(alphaR)-alpha-((2S,5R)-4-allyl-2,5-dime thyl-1-piperazinyl)-3-methoxybenzyl]-N,N-diethylbenzamide (SNC-80) compared to saline-treated groups. Under these conditions, repeated s.c. injection of (-)U-50,488H significantly enhanced both mu- and delta-opioid receptor agonist-stimulated [35S]GTPgammaS binding in the membrane of the thalamus. On the contrary, either repeated administration of morphine (s.c. or i.c.v.) or SNC-80 failed to affect the kappa-opioid receptor agonist-induced antinociception and G-protein activation. Taken together, these results suggest that repeated stimulation of kappa-opioid receptor markedly increases the functional mu- and delta-opioid receptors, whereas repeated stimulation of either mu- or delta-opioid receptor had no direct effect on kappa-opioidergic function in mice.

The gamma-aminobutyric acid A (GABA(A)) receptor subunit expression of the dopaminergic cells of the substantia nigra (SN) was investigated in the present study. Especially the dopaminergic cells, located in the pars compacta of SN (SNc), are of great neurologic interest, because the functional deficit and depletion of these cells are the correlate of Parkinson's disease. We used a combination of in situ hybridization histochemistry (ISH) and immunohistochemistry (IHC) on sections of human postmortem mesencephalon to investigate the expression of GABA(A) receptor subunit messenger RNAs (mRNAs) and of the receptor protein in dopaminergic SN cells. Immunohistochemical detection of tyrosine hydroxylase (TH), the pivotal enzyme of dopamine synthesis, was used to define the boundaries of SN pars reticulata (SNr) and pars compacta subregions. In SNr, all neurons were labeled by subunit-specific oligonucleotide probes and the amount of GABA(A) receptor mRNA expression was quantified as alpha(1) = beta(2)>> gamma(2)>> alpha(3). In contrast, in SNc, only around 25% of neurons expressed mRNA transcripts of GABA(A) receptor subunits, quantified as alpha(1) = beta(2)>> gamma(2)>> alpha(3)>> alpha(4) = beta(3). In approximately the same percentage of neurons, which were labeled by alpha(1)-subunit-specific probe, the alpha(1)-subunit also was detected at the protein level by a specific monoclonal antibody. We, therefore, could demonstrate that a subset of dopaminergic neurons in human SNc receive inhibitory synaptic input by means of GABA(A) receptors mainly of the alpha(1)beta(2)gamma(2) subtype. This might represent a negative feedback loop between the striatum and the SNc and be a target of pharmacologic interventions in neurodegenerative diseases such as Parkinson's disease.

Opioids alter immune function by binding to opioid receptors on cells of the immune system, or indirectly by acting on receptors within the central nervous system. Mu-selective opioid agonists are generally associated with immunosuppression, whereas delta-opioid receptor-selective agonists are commonly associated with immunopotentiation. We have previously shown that intracerebroventricular administration of the nonpeptide delta-opioid receptor agonist (+)-4-((alpha R)-alpha-((2S, 5R)-4-allyl-2, 5-dimethyl-1-piperazinyl)-3-methoxybenzyl)-N, N-diethyl-benzamide (SNC 80) did not alter certain parameters of immunocompetence. In the present study, we studied the in vitro and ex vivo effects of SNC 80 on rat macrophage and lymphocyte functions. We showed that SNC 80 at concentrations of 10(-7) M and 10(-6) M, significantly (P < 0.01) stimulated the in vitro production of tumor necrosis factor-alpha (TNF-alpha) (60-100% increase) and nitric oxide (34-67% increase) by resident and LPS-stimulated peritoneal macrophages. Similarly, intravenous administration of SNC 80 (6.8 mg/kg) significantly (P < 0.01) increased the production of TNF-alpha and nitric oxide (2- and 1.5-fold increases respectively, compared with saline-injected control) by LPS-stimulated splenic macrophages. In addition, intravenous injection of SNC 80 plus Con A potentiated ex vivo LPS-stimulated macrophage functions. SNC 80 could potentially be utilized in various clinical situations where immunosuppression is undesirable.

Despite universal use of opioids in the clinic to inhibit pain, there is relatively little known of their peripheral actions on sensory nerve endings, where in fact they may be better targeted with more widespread applications. Here we show differential effects of mu-, kappa-, and delta-opioids on mechanosensitive ferret esophageal vagal afferent endings investigated in vitro. The effects of selective agonists [d-Ala(2),N-Me-Phe(4),Gly-ol(5)]-enkephalin (DAMGO), 2-(3, 4-dichlorophenyl)-N-methyl-N-[(1S)-1phenyl-2-(1-pyrrolidinyl) ethyl] acetamide hydrochlorine (ICI 199441), and (+)-4-[(alphaR)-alpha-((2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl)-3-methoxybenzyl]-N,N-diethylbenzamide (SNC-80), respectively, on mechanosensory stimulus-response functions were quantified. DAMGO (10(-7) to 10(-5) M) reduced the responses of tension receptors to circumferential tension (1-5 g) by up to 50%, and the responses of mucosal receptors to mucosal stroking (10-1,000 mg von Frey hair) by >50%. DAMGO effects were reversed by naloxone (10(-5) M). Tension/mucosal (TM) receptor responses to tension and stroking were unaffected by DAMGO. ICI 199441 (10(-6) to 10(-5) M) potently inhibited all responses except TM receptor responses to tension, and SNC-80 (10(-5) to 10(-3) M) had no effect other than a minor inhibition of mucosal receptor responses to intense stimuli at 10(-3) M. We conclude that mu- and kappa-opioids have potent and selective peripheral effects on esophageal vagal afferents that may have applications in treatment of disorders of visceral sensation.

Degeneration of dopamine neurons in the substantia nigra pars compacta (SNc) plays an important role in the pathophysiology of neurodegenerative diseases like Parkinsonism and vascular dementia. SNc dopamine neurons both in vitro and in vivo show sensitivity to hypoxic/ischemic conditions and undergo degeneration. In acute brain slices, these dopamine neurons undergo hyperpolarization during hypoxia and hypoglycemia, which results in silencing of the neurons. However, the role that SNc excitatory synapses play in this process is poorly understood. Here we examined the effect of oxygen/glucose deprivation (OGD) on glutamatergic synaptic transmission in the SNc in a rat midbrain slice preparation. OGD for 5 min caused pre-synaptic ischemic long-term depression (iLTD) of glutamate transmission, as both alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid- and N-methyl-D-aspartate receptor-mediated synaptic currents in SNc dopamine neurons were depressed to a similar extent. This depression began immediately after exposure to OGD and was not recovered upon washout of OGD. Pharmacological studies revealed that the iLTD was triggered by a rise in post-synaptic intracellular calcium and mediated by activation of pre-synaptic adenosine A(1) receptors, which reduced glutamate-dependent synaptic transmission by activating ATP-dependent potassium channels. Furthermore, we observed that iLTD did not occlude tetanic long-term depression (LTD) at the SNc excitatory synapses, suggesting that these two forms of LTD involve different pathways. Taken together, our results showed that brief exposure to hypoxia and hypoglycemia results in LTD of synaptic activity at glutamatergic synapses onto SNc neurons and this phenomenon could represent a protective mechanism by reducing ischemia-induced excitotoxic injury to dopamine neurons.

We evaluated the anti-exudative effects (Evan's blue) of mu-, delta- and kappa-opioid receptor agonists in a rat model of carrageenan-induced acute inflammation. The contribution of different components was assessed after the administration of: cyclosporine A, capsaicin, 6-hydroxydopamine, compound 48/80, and specific histamine-receptor antagonists. The results show that the mu-opioid receptor agonists morphine and fentanyl and the delta-opioid receptor agonists DPDPE (enkephalin, [D-Pen(2,5)]) and SNC 80 ((+)-4-[(alpha R)-alpha((2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl)-3-methoxybenzyl]-N,N diethylbenzamide) decrease plasma extravasation in a dose-dependent manner, with a biphasic response. The effects were reversed by specific antagonists, and are predominantly mediated by peripheral opioid receptors. The integrity of sensory and sympathetic fibres is essential for the anti-exudative effects of fentanyl and DPDPE. Histamine and functional histamine H(2) and H(3) receptors are required for morphine and fentanyl (but not DPDPE) inhibition of plasma extravasation, suggesting different mechanism for mu- and delta-opioid receptor agonists. The present findings implicate multiple sites and mechanisms in the anti-exudative effects of exogenous opioids.

OBJECTIVE

The present study aimed to demonstrate protective effects of alpha lipoic acid on experimental sciatic nerve crush injury model assessed with functional and electronmicroscopy analyses.

METHODS

In this study, groups were; Group 1; sham operated, Group 2; applied only sciatic nerve crush (Control), Group 3; Sciatic nerve crush + treated ALA 25 mg/kg (received orally) and Group 4; Sciatic nerve crush + treated ALA 50 mg/kg. Subsequently, sciatic nerves crush injury induced by forceps. At the second and fourth week, all animals were evaluated for sciatic functional index (SFI) and histomorphometric analyses with electronmicroscopy.

RESULTS

The SFI was significantly increased for both ALA-treated groups 30 days post-injury compared with control groups. The elecronmicroscopy results demonstrated that the axon diameter, the myelin diameter, the area of regenerating axon and miyelin were better in the treatment group than in the control group. Also ALA decreased IL-1β and Caspase 3 levels that increased in SNC group.

CONCLUSIONS

These results suggest that ALA neuroprotective agent for peripheral nerve injury (PNI) and promoted peripheral nerve regeneration via its anti-inflammatory and antiapoptotic effects.

Dopaminergic cell death in the ventral and dorsal tiers of substantia nigra pars copmacta (SNc) and their prevention by anti-oxidant diet was immunohistochemically studied in the zitter mutant rats, which are characterized by abnormal metabolism of superoxide. Similar to previous reports, the number of SNc neurons in Nissl-stained section decreased with age. Tyrosine hydroxylase (TH) immunohistochemistry demonstrated that the dopaminergic neurons in the ventral tier of SNc degenerated early, whereas the dorsal tier gradually degenerated with age. Thus, the ventral tier dopaminergic neurons are affected first, but the dorsal tier neurons do become impact by the zi/zi mutation. Following 9-month period after weaning, zitter rats supplemented with 500 mg D,L-alpha-tocophenol (VE(+))/kg diet exhibited a significant increased of surviving TH-immunoreactive neurons in both the tiers of SNc as compared with the zi/zi rats with control and VE(-) diets. These results suggest that VE supplement may slow the dopaminergic cell loss in zitter mutant rat, and further support that degeneration of the dopaminergic neurons in this mutant rat is caused by oxidant stress. Thus, the zitter rat may represent a good model for studying the dopaminergic cell death by superoxide species.

Delta- and kappa-opioid receptors (OPRs), but not micro-OPRs, are expressed in the myenteric plexus of the porcine distal small intestine. In a subpopulation of myenteric neurons, delta- and kappa-OPRs seem to be colocalized and may functionally interact. In this study, radioligand binding was used to characterize myenteric OPR populations in detail. The nonselective OPR antagonist [3H]diprenorphine bound to a single, high-affinity site in myenteric neural membrane homogenates. Naloxone displaced 65 and 59% of [3H]diprenorphine binding from this site in Na(+)-free Tris and Krebs-HEPES buffers, respectively. Naltrexone-derived delta- and kappa-OPR antagonists, including naltriben, 7-benzylidenenaltrexone, nor-binaltorphimine, and 5'-guanidinonaltrindole, displaced [3H]diprenorphine from two distinct binding sites to levels similar to that of naloxone. The selective delta-OPR ligands Tyr-1,2,3,4-tetrahydroisoquinoline-Phe-Phe-OH (TIPP), [D-Pen2,D-Pen5]enkephalin (DPDPE), [D-Ala2, Glu4]deltorphin II, and (+)-4-[(alphaR)-alpha((2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl-3-methoxybenzyl)-N,N-diethylbenzamide (SNC-80) and the kappa-OPR agonist (D-(5alpha,7alpha,8beta)-(-)-N-methyl-N-(7-(1-pyrrolidinyl)-1-oxoaspiro-(4,5)dec-8-yl) benzeneacetamide (U-69,593) displaced [3H]diprenorphine from three independent binding sites; these included high-affinity delta- and kappa-OPR sites, and a residual binding site. Residual [3H]diprenorphine binding was displaced by the selective kappa-OPR antagonist nor-binaltorphimine after saturation of delta and kappa sites, respectively, with DPDPE and U-69,593. The residual binding site displayed low affinity for delta- and kappa-OPR agonists and TIPP, as well as moderate affinity for naltrexone-derived ligands, properties reminiscent of delta-/kappa-OPR heterodimers.

Neonatal cattle and in part neonates of other species have manyfold higher plasma concentrations of nitrite plus nitrate than mature cows and subjects of other species, suggesting an enhanced and needed activation of the nitric oxide (NO) axis at birth. While the biological half-life of NO is short (<1 sec), its functionality can be prolonged, and in many regards more discretely modulated, when it reacts with low-molecular-weight and protein-bound thiols to form S-nitrosothiols (RSNO), from which NO subsequently can be rereleased. We used the calf as a model to test the hypothesis that plasma concentrations of RSNO are elevated at birth in mammals, correlate with ascorbate and urate levels, are selectively generated in critical tissue beds, and are generated in a manner temporally coincident with changes in tissue levels of active NO synthases (NOS). Plasma concentrations of RSNO, ascorbate, and urate were highest immediately after birth (Day 0), dropped >50% on Day 1, and gradually decreased over time, reaching a nadir in mature cattle. Albumin and immunoglobulin G were identified as major plasma RSNO. The presence of S-nitrosocysteine (SNC, a validated marker for S-nitrosylated proteins), inducible NOS (iNOS), and activated endothelial NOS (eNOS phosphorylated at Ser1177) in different tissues was analyzed by immunohistochemistry in another group of similar-aged calves. SNC, iNOS, and phosphorylated eNOS were detected in liver and ileum at the earliest timepoint of sampling (4 hrs after birth), increased between 4 and 24 hrs, and then declined to near-nondetectable levels by 2 weeks of life. Our data show that the neonatal period in the bovine species is characterized by highly elevated and coordinated NO-generating and nitrosylation events, with the ontogenetic changes occurring in iNOS and eNOS contents in key tissues as well as RSNO products and associated antioxidant markers.

Parkinson's disease (PD) is characterized by the destruction of dopaminergic cells in the substantia nigra (SN). The cause of the cell death and the development of Parkinsonism is however unknown. There are increasing evidences to suggest the involvement of glutamate mediated by its receptors. Using immunohistochemistry and cell counting, the present study investigated whether the numbers of neurons immunostained with glutamate alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor subunits GluR1, GluR2/3 and GluR4 in the SN of rats would change after the injection of 6-hydroxydopamine (6-OHDA) into the SN. The results showed that the numbers of GluR1 positive cells were significantly decreased in the substantia nigra pars compacta (SNc), pars reticulata (SNr) and pars lateralis (SNl) from 3 days (13.7%) to 3 months (40.3%) and of GluR2/3 cells, from 1 week (17.6%) to 3 months (19.1%) after 6-OHDA injection, compared to those in the contralateral non-injected side. There was, however, no significant difference in the number of GluR4 positive cells between the injected and non-injected SN. The results were discussed.

Bilateral microinjections of an excitatory amino acid, alpha-kainate (KA), 5-50 ng, into the substantia nigra pars reticulata (SNR) result in an increase in the muscle tone and catalepsy in rats. The preferential KA/quisqualate antagonist, gamma-D-glutamylaminomethylsulphonate (gamma-D-GAMS), 10 micrograms, blocks the actions of KA, 25 ng, when coadministered into the SNR. The chemical lesion of the caudate-putamen with 6-hydroxydopamine (6-OHDA) does not affect either increases in the muscle tone or catalepsy produced by KA, 25 ng, from the SNR. The lesion of the caudate-putamen with ibotenate moderately enhances the effect of KA, 25 ng, on the muscle tone. Microinjections of KA, 25 ng, into the substantia nigra pars compacta (SNC) do not increase the muscle tone and lead to significantly less pronounced catalepsy relative to that observed following the injections of KA into the SNR. Unilateral microinjections of KA, 10-50 ng, into the SNR elicit ipsilateral turning in rats in a dose- and time-dependent manner. Unilateral application of gamma-D-GAMS, 1-10 micrograms, into the SNR produces contralateral turning. The turning evoked by KA, 25 ng, or gamma-D-GAMS, 10 micrograms, is affected neither by 6-OHDA nor by ibotenate lesion of the caudate-putamen. These results demonstrate that excitatory neurotransmission in the substantia nigra participates in the regulation of the muscle tone and posture in rats.

BACKGROUND

Successive negative contrast (SNC) describes a change in the behaviour of an animal following a downshift in the quantitative or qualitative value of an expected reward. This behavioural response has been hypothesised to be linked to affective state, with negative states associated with larger and/or prolonged shifts in behaviour.

OBJECTIVE

This study has investigated whether different psychopharmacological treatments have dissociable actions on the SNC effect in rats and related these findings to their actions on different neurotransmitter systems and affective state.

METHODS

Animals were trained to perform a nose-poke response to obtain a high-value food reward (four pellets). SNC was quantified during devalue sessions in which the reward was reduced to one pellet. Using a within-subject study design, the effects of acute treatment with anxiolytic, anxiogenic, antidepressant and dopaminergic drugs were investigated during both baseline (four pellets) or devalue sessions (one pellet).

RESULTS

The indirect dopamine agonist, amphetamine, attenuated the SNC effect whilst the D1/D2 antagonist, alpha-flupenthixol, potentiated it. The antidepressant citalopram, anxiolytic buspirone and anxiogenic FG7142 had no specific effects on SNC, although FG7142 induced general impairments at higher doses. The α2-adrenoceptor antagonist, yohimbine, increased premature responding but had no specific effect on SNC. Results for the anxiolytic diazepam were mixed with one group showing an attenuation of the SNC effect whilst the other showed no effect.

CONCLUSIONS

These data suggest that the SNC effect is mediated, at least in part, by dopamine signalling. The SNC effect may also be attenuated by benzodiazepine anxiolytics.

Opioids like morphine, represent a major source of relief for most chronic moderate to severe nonmalignant pain. However, opioid abuse may lead to infections such as hepatitis and AIDS because opioids have been associated with suppressing various parameters of immune function including antimicrobial resistance, antibody production, monocyte-mediated phagocytosis, and both neutrophil and monocyte chemotaxis. We have previously reported immunopotentiating properties of non-peptidic opioid receptor selective agonists and antagonists. In this study, we evaluated the effects of the nonpeptidic delta-opioid receptor agonist (+)-4-((alpha R)-alpha-((2S, 5R)-4-allyl-2, 5-dimethyl-1-piperazinyl)-3-methoxybenzyl)-N, N-diethyl-benzamide (SNC 80) on chemotaxis of rat thymic and human peripheral blood mononuclear cells by using a modified Wilkinson chamber. Cell recruitment is an essential process in acute and chronic inflammatory responses. We observed that SNC 80 at concentrations of 10(-10), 10(-9), 10(-8), 10(-7), and 10(-6) M, significantly (p < 0.01) stimulated rat thymic (1.3, 1.55, 1.58, 1.75, and 1.8-fold increases respectively) and human leukocyte (1.13, 1.37, 1.43, 1.7, 1.83 fold-increases respectively) chemotaxis (demonstrated by checkerboard assays), compared with untreated control. The effects of SNC 80 on chemotaxis of rat and human leukocytes were antagonized by naloxone, indicating that the modulation of chemotaxis by SNC 80 is via a classic opioid receptor. The development and use of non-peptidic opioids like SNC 80 could have an immediate impact not only as potent analgesics, but in immunoregulation.

Quantitative autoradiography using [3H]-SCH 23390, [3H]-sulpiride and [3H]-forskolin was used to assess the effects of single and combined neurotoxin lesions of the nigrostriatal pathway in the rat brain on dopamine (DA) receptor subtypes and adenylate cyclase (AC), respectively. Ibotenic acid (IA) lesions of the caudate-putamen (CPu) resulted in near total loss of both [3H]-SCH 23390 and of [3H]-forskolin binding in the ipsilateral CPu and substantia nigra reticulata (SNR). [3H]-sulpiride binding in the CPu was only partially removed by this same lesion, and nigral [3H]-sulpiride binding was virtually unchanged. 6-Hydroxydopamine (6-OHDA) and IA lesions of the substantia nigra compacta (SNC) did not affect [3H]-SCH 23390 or [3H]-forskolin binding, but largely removed [3H]-sulpiride binding in the SNC. A 6-OHDA lesion of the nigrostriatal pathway followed by an ipsilateral IA injection of the CPu failed to further reduce [3H]-sulpiride binding in the CPu. These results demonstrate that postsynaptic DA receptors in the CPu are of both the D1 and D2 variety; however, a portion of D2 receptors in the CPu may be presynaptic on afferent nerve terminals to this structure. D1 receptors in the SNR are presynaptic on striatonigral terminals, whereas the D2 receptors of the SNC are autoreceptors on nigral DA neurons. The existence of presynaptic D2 receptors on nigrostriatal DA-ergic terminals could not be confirmed by this study. Co-localization of D1 receptors and AC occurs in both the CPu and SNR.

The effects of nitric oxide (NO) and other cysteine modifying agents were examined on cyclic nucleotide-gated (CNG) cation channels from rat olfactory receptor neurons. The NO compounds, S-nitroso-cysteine (SNC) and 3-morpholino-sydnonomine (SIN-1), did not activate the channels when applied for up to 10 min. The cysteine alkylating agent, N-ethylmaleimide (NEM), and the oxidising agent, dithionitrobensoate (DTNB), were also without agonist efficacy. Neither SNC nor DTNB altered the cAMP sensitivity of the channels. However, 2-min applications of SIN-1, SNC and DTNB inhibited the cAMP-gated current to approximately 50% of the control current level. This inhibition showed no spontaneous reversal for 5 min but was completely reversed by a 2-min exposure to DTT. The presence of cAMP protected the channels against NO-induced inhibition. These results indicate that inhibition is caused by S-nitrosylation of neighboring sulfhydryl groups leading to sulfhydryl bond formation. This reaction is favored in the closed channel state. Since recombinantly expressed rat olfactory alpha and beta CNG channel homomers and alpha/beta heteromers are activated and not inhibited by cysteine modification, the results of this study imply the existence of a novel subunit or tightly bound factor which dominates the effect of cysteine modification in the native channels. As CNG channels provide a pathway for calcum influx, the results may also have important implications for the physiological role of NO in mammalian olfactory receptor neurons.

Using acutely dissociated substantia nigra pars compacta (SNC) dopaminergic (DA) neurons, our previous studies indicated that neurotensin (NT) excites SNC DA neurons by increasing the cationic conductance and reducing the inwardly rectifying K+ conductance. Further investigation also revealed that pertussis toxin (PTX)- insensitive G-proteins mediate neurotensin modulation of cation and potassium channels. G alpha q and G alpha 11 are widely distributed in various tissues including the brain and likely to mediate PTX-insensitive signal transductions in the nervous system. In this study, two different experiments were conducted to test the hypothesis that G alpha q/11 mediates neurotensin regulation of the cationic and K+ conductances. First, we investigated the expression of G alpha q and G alpha 11 mRNAs in NT-responsive SNC DA neurons by combining whole-cell patch-clamp recordings with single-cell reverse transcriptase-polymerase chain reaction (RT-PCR) assay. After recording NT-evoked membrane currents, the cellular content was harvested from single neurons and used as the template for the subsequent RT-PCR analysis. Both G alpha q and G alpha 11 mRNAs were present in all SNC DA neurons that responded to neurotensin. SNC DA neurons were also internally dialyzed with an antibody directed against the common C-terminus of G alpha q and G alpha 11 during whole-cell recordings. In DA neurons perfused with the anti-G alpha q/11 antiserum, neurotensin failed to evoke inward currents resulting from the opening of cation channels and the closure of inward rectifier K+ channels. It is concluded that NT modulation of cation and inward rectifier K+ channels in SNC DA neurons is transduced by G alpha q and/or G alpha 11.

Iresine herbstii Hook. (Amaranthaceae) and Brugmansia arborea (L.) Lagerheim (Solanaceae) are used in the northern Peruvian Andes for magic-therapeutical purposes. The traditional healers use Iresine herbstii with the ritual aim to expel bad spirits from the body. Furthermore, Iresine herbstii was used in association with other plants, such as Trichocereus pachanoi Britt. et Rose, for divination, to diagnose diseases, and to take possession of another identity. Also, species of Brugmansia have been reported to be used during ritual practices for magical and curative purposes. Given the above evidence, the aim of the present study is to evaluate if the central effects of Iresine herbstii and Brugmansia arborea could be associated with interaction with SNC receptors. Two Iresine herbstii extracts (methanolic and aqueous) and one Brugmansia arborea aqueous extract were tested for in vitro affinity on 5-HT(1A), 5-HT(2A), 5-HT(2C), D1, D2, alpha(1), and alpha(2) receptors by radioligand binding assays. The biological materials for binding assay (cerebral cortex) were taken from male Sprague-Dawley rats. The extracts affinity for receptors is definite as inhibition percentage of radioligand/receptor binding and measured as the radioactivity of remaining complex radioligand/receptor. The data obtained for Iresine extracts have shown a low affinity for the 5-HT(1A) receptor and no affinity for 5-HT(2A) receptor. Otherwise the methanolic extract showed affinity for 5-HT(2C) receptor (IC(50): 34.78 microg/ml) and for D1 receptor (IC(50): 19.63 microg/ml), instead the Iresine aqueous extract displayed a lower affinity for D1 (48.3% at the maximum concentration tested) and a higher value of affinity for D2 receptors (IC(50): 32.08 microg/ml). The Brugmansia aqueous extract displayed affinity for D1 receptors (IC(50): 17.68 microg/ml), D2 receptors (IC(50): 15.95 microg/ml) and weak affinity for the serotoninergic receptors. None of the three extracts showed relevant affinity to the alpha(1), and alpha(2) receptors. The results of our experiments indicate that Iresine herbstii methanolic extract was able to interact with the central 5-HT(2C) and D1 receptors and Iresine herbstii aqueous extract showed affinity for D2 receptors, thus confirming their ritual use. Instead Brugmansia arborea was able to interact only with the central dopamine receptors tested. Parallel studies are currently in progress for evaluating the extracts affinity and active components towards these and other receptor types (GABAergic).

Gamma-aminobutyric acid(A) (GABA(A)) receptors (GABA(A)R) are inhibitory heteropentameric chloride ion channels comprising a variety of subunits and are localized at postsynaptic sites within the central nervous system. In this study we present the first detailed immunohistochemical investigation on the regional, cellular, and subcellular localisation of alpha(1), alpha(2), alpha(3), beta(2,3), and gamma(2) subunits of the GABA(A)R in the human substantia nigra (SN). The SN comprises two major regions, the SN pars compacta (SNc) consisting of dopaminergic projection neurons, and the SN pars reticulata (SNr) consisting of GABAergic parvalbumin-positive projection neurons. The results of our single- and double-labeling studies demonstrate that in the SNr GABA(A) receptors contain alpha(1), alpha(3), beta(2,3), and gamma(2) subunits and are localized in a weblike network over the cell soma, dendrites, and spines of SNr parvalbumin-positive nonpigmented neurons. By contrast, GABA(A)Rs on the SNc dopaminergic pigmented neurons contain predominantly alpha(3) and gamma(2) subunits; however there is GABA(A)R heterogeneity in the SNc, with a small subpopulation (6.5%) of pigmented SNc neurons additionally containing alpha(1) and beta(2,3) GABA(A)R subunits. Also, in the SNr, parvalbumin-positive terminals are adjacent to GABA(A)R on the soma and proximal dendrites of SNr neurons, whereas linear arrangements of substance P-positive terminals are adjacent to GABA(A) receptors on all regions of the dendritic tree. These results show marked GABA(A)R subunit hetereogeneity in the SN, suggesting that GABA exerts quite different effects on pars compacta and pars reticulata neurons in the human SN via GABA(A) receptors of different subunit configurations.