Differential behavioral effects induced by intrathecal microinjection of opiates: Comparison of convulsive and cataleptic effects produced by morphine, methadone, andd-Ala-methionine-enkephalinamide

Intrathecal (i.t.) microinjection of 400 μg of morphine in rats induces convulsive activity restricted to the hindlimbs. This activity is potentiated rather than antagonized by naltrexone, is potentiated in animals pretreated over 6 days with increasingly higher systemic doses of morphine, and is potentiated and prolonged by high thoracic spinalization. Similar to morphine, convulsive spinal activity could also be elicited with i.t. injection of the non-opiate convulsant penthylenetetrazol (PTZ). However, methadone, d-ala²-methionine-enkephalinamide (DALA), or naltrexone, injected i.t. at equimolar doses with morphine did not produce similar convulsive behavior. DALA and methadone rather produced pronounced spinal catalepsy. It was concluded: (1) that the spinal convulsive action of morphine is not mediated by specific opiate receptors; and (2) that the spinal cord may be one of the sites where high doses of systemically administered morphine may produce convulsions.     

Cataleptic effects of opiates following intrathecal administration

Intrathecal (i.t.) administration of D-ala2-methionine-enkephalinamide (DALA) and methadone induces pronounced hindlimb catalepsy in rats. DALA is more potent than methadone in inducing this behavior and catalepsy induced by both compounds is partially blocked by prior treatment with naltrexone. Intrathecal morphine induces convulsive hindlimb activity at doses of 25, 100 and 400 micrograms. However, only the highest dose produces indications of cataleptic behavior. It is suggested that whereas the convulsive behavior induced by i.t. morphine is not mediated by specific opiate receptors, catalepsy induced by all 3 compounds is.     

Relationship between resting cytosolic Ca and responses induced byN-methyl-d-aspartate in hippocampal neurons

Cytosolic calcium concentrations ([Ca²⁺]_i) in cultured hippocampal neurons from rat embryos were measured using fura-2. Neurons with higher resting [Ca²⁺]_i showed greater [Ca²⁺]_i responses toN-methyl-d-aspartate (NMDA) and K⁺ depolarization. There was a strong relationship between resting [Ca²⁺]_i and the maximal changes in [Ca²⁺]_i (Δ[Ca²⁺]_i), which fit the our proposed equation to describe this relationship.     

Decrease in δ-opioid receptor density in rat brain after chronic [d-Ala,d-Leu]enkephalin treatment

Chronic treatment of Sprague-Dawley rats with [d-Ala²,d-Leu⁵]enkephalin (DADLE) resulted in the development of tolerance to the antinociceptive effect of this opioid peptide. When opioid receptor binding was measured, time-dependent decreases in [³H]diprenorphine binding to the P₂ membranes prepared from the cortex, midbrain and striatum were observed. Scatchard analysis of the saturation binding data revealed a decrease in B_max values and no change in the K_d values of [³H]diprenorphine binding to these brain regions, indicative of down-regulation of the receptor. This reduction in the opioid receptor binding activities could be demonstrated to be due to the DADLE effect on the δ-opioid receptors in these brain regions. When [³H]DADLE binding was carried out in the presence of morphiceptin, a significant reduction in the δ-opioid receptor binding was observed in all brain areas tested. μ-Opioid receptor binding decrease was observed only in the striatum after 5 days of DADLE treatment. Additionally, the onset of δ-opioid receptor decrease in the midbrain area was rapid, within 6 h of the initiation of the chronic DADLE treatment. Thus, analogous to previous observations in which chronic etorphine treatment preferentially reduced μ-opioid receptor binding, chronic DADLE treatment preferentially reduced δ-opioid receptor binding activity.     

Blockade ofCa influx through theN-methyl-d-aspartate receptor ion channel by the non-psychoactive cannabinoid HU-211

The effects of the synthetic non-psychoactive cannabinoid(+)-(3S,4S)-7-hydroxy-Δ⁶-tetrahydrocannabinol 1,1-dimethylheptyl (HU-211) on the activity of theN-methyl-d-aspartate (NMDA) receptor/ion channel were examined. HU-211 non-competitively blocks the increase in binding of[³H]N-[1-(2-thienyl)-cyclohexyl]piperidine ([³H]TCP) induced by the polyamines spermine and spermidine or by glutamate and glycine. HU-211 does not, however, affect the direct binding of [³H]glycine and [³H]glutamate to their binding sites on the NMDA receptor, which suggests that the effects of HU-211 are not mediated via the binding sites of glutamate-, glycine- and phencyclidine-like drugs or of polyamines. HU-211 can also block⁴⁵Ca²⁺ uptake through the NMDA-receptor/ion channel in primary cell cultures of rat forebrain. All of the above inhibitory effects of HU-211 on the NMDA-receptor/ion channel activity are stereospecific, since the(−)(3R,4R) enantiomer (HU-210) is ineffective.     

Molecular and functional characterization of Xenopus laevis N-methyl-d-aspartate receptors

N-methyl-d-aspartate (NMDA) receptors of many different vertebrates have been characterized in the past. However, little information is available about amphibian NMDA receptors. Here, we investigated the South African clawed frog Xenopus laevis NR1 subunit at the molecular and functional level. In this subunit, which is obligatory for functional NMDA receptor complexes, we found three exons, the N1, C1, and C3 cassettes, being alternatively spliced. Combinations of these cassettes generated six different splice variants, which were functionally characterized in oocytes. The Xenopus NR1 isoforms generally showed the same functional properties as their mammalian homologs when coexpressed with rat NR2B. In coexpression with Xenopus NR2B, however, some properties changed significantly. This included a Zn²⁺-mediated potentiation of current amplitudes for some subunit combinations which lasted for several minutes. This mechanism presents a novel form of Xenopus NMDA receptor modulation, possibly mediating a form of short-term potentiation in the Xenopus central nervous system.     

Simultaneous measurement of cholecystokinin- and vasoactive intestinal polypeptide-like immunoreactivity from cat frontal cortex in vitro: Effect of morphine andd-Ala-d-Leu-Enkephalin

The two peptides vasoactive intestinal polypeptide (VIP) and cholecystokinin (CCK) have been demonstrated to be discretely distributed in the cerebral cortex. This distribution closely parallels the distribution of μ- and δ-opiate receptors in the frontal cortex. The basal efflux and potassium-stimulated release of VIP- and CCK-immunoreactivity was studied in the presence and absence of morphine andd-Ala²-d-Leu⁵-enkephalin (DADL), agents with relative affinity for the μ and δ receptors, respectively. The basal efflux of VIP- and CCK-immunoreactivity was not affected by these opiate; however, the potassium-stimulated release of VIP-immunoreactivity was profoundly inhibited in a dose-dependent manner by both morphine (ED₅₀ = 1 × 10⁻⁹M) and DADL (ED₅₀= 3.02 × 10⁻⁹M). The inhibition produced by either morphine or DADL was shown to be reversed by naloxone.     

Inhibition of nitric oxide formation does not protect murine cortical cell cultures from N-methyl-d-aspartate neurotoxicity

We examined the role of nitric oxide in N-methyl-d-aspartate (NMDA) receptor-mediated neurotoxicity in rat and mouse primary cortical cell cultures. In rat and mouse cultures, the NO synthase inhibitor, N^G-Nitro-l-arginine, blocked cGMP formation but not neuronal cell death following a 5–10 min exposure to 300–500 μM NMDA. N^G-Monomethyl-l-arginine was also unable to prevent neuronal death. In contrast, the non-competitive NMDA receptor antagonist, dextrophan, prevented both cGMP formation and cell death. While other data suggest that the synthesis of nitric oxide can mediate NMDA receptor-mediated neurotoxicity, present results suggest that such synthesis is not necessarily required.     

Contraversive circling induced by ventral tegmental microinjections of moderate doses of morphine and [d-Pen,d-Pen]enkephalin

Unilateral ventral tegmental area (VTA) injections of morphine and [d-Pen²,d-Pen⁵]enkephalin (DPDPE), caused contraversive circling at doses of 1.2, 12, and 24 nmol. Similar doses of the selective κ-agonist U-50,488H were ineffective. These data suggest a common mechanism for the circling, locomotion and facilitation of brain stimulation reward caused by VTA morphine, and distinguish this mechanism for that of feeding which is caused by both morphine and κ-actions in this region.     

Insulin increases entrane of 2-deoxy-d-[H]glucose in isolated rat brain microvessels

Insulin in a dose of 0.5 U ml⁻¹ in the incubation medium did not change ATP and phosphocreatine contents in the isolated rat brain microvessels, when energy production was supported by 5.5 mmol l⁻¹ glucose. However, the entrance of non-metabolizable glucose analogue, 2-deoxy-d-[³H]glucose into the microvessel cells was strongly enhanced, almost 18-fold.     

Determination of F-fluoro-2-deoxy-d-glucose rate constants in the anesthetized baboon brain with dynamic positron tomography

We have determined the rate constants (k_i^*) of ¹⁸F-fluorodeoxyglucose (FDG) in the unlesioned baboon brain, for use in positron emission tomography (PET) measurements of glucose utilization. In contrast to earlier reports, we used a radiosynthesis which garantees production of FDG essentially uncontaminated by fluorodeoxymannose, and an improved determination of k_i^* by (1) direct measurement of the time-shift between bolus arrival in femoral arterial plasma and brain, (2) rapid initial PET frames, and (3) extended data acquisition (up to 180 min). Young adult baboons were studied under anesthesia with either phencyclidine or etomidate. The FDG time-activity curves obtained from temporal grey matter showed a consistent decline after about 80 min, indicating true product loss. Three-compartment modelling was performed for increasing fitting intervals (20–120 min) with both a 5-parameter (K₁^* − k₄^*, and vascular volume (V_o)) and a 4-parameter (K₁^* − k₃^*, V_o) model. With the latter, both the calculated FDG net clearance ((K^* = K₁^* · k₃^*/(k₂^* + k₃^*)) and the fitted kinetic constants were dependent on fitting interval, i.e., they showed sustained unstability. With the former, the constant k₄^*, which presumably represents dephosphorylation, was overestimated and unstable for short fitting times (presumably due to heterogeneous brain compartments in the sample tissue), but stabilized at − 0.01 min⁻¹ for fitting times ≥80 min; K₁^* − k₃^* and K^* were also stable after this time. These findings were identical for both anesthetic regimen. Thus, in the anesthetized baboon, the FDG k₁^* values can be reliably determined based on an adequate PET acquisition paradigm and with a model that incorporates k₄^* and ≥80 min time-activity data.     

Stimulation of the N-methyl-d-aspartate receptor promotes the biochemical differentiation of cerebellar granule neurons and not astrocytes

Cerebellar granule cells are believed to be glutamatergic, but, as they receive excitatory amino acidergic input from mossy fibers, they also possess N-methyl-d-aspartate (NMDA) receptors. The possible involvement of these NMDA receptors in the biochemical differentiation of cultured granule neurons was studied in terms of the specific activity of phosphate-activated glutaminase, an enzyme important in the synthesis of the putative neurotransmitter pool of glutamate. When the partially depolarized cells were treated with NMDA for the last 3 days (i.e. between 2 and 5 days in vitro), it elevated specific activity of glutaminase in the dose- and time-dependent manners. The half-maximal effect was obtained at about 10 μM NMDA, whereas the maximum concentration, which produced about a 2.7-fold increase in 5-day-old cultures, was about 50 μM NMDA. This increase in glutaminase was completely blocked by the NMDA receptor antagonist, 2-amino-5-phosphonovaleric acid, and by the NMDA receptor-linked Ca²⁺ ion channel blockers, MK 801 and Mg²⁺. The effect of NMDA was not related to the survival of the granule cells, as the experiments were carried out before the dependence on high K⁺ for the survival of granule cells develops in culture, and during the period of investigation none of the compounds used compromised the survival of these cells. The enhancement of glutaminase activity was due to an induction in enzyme protein, since it was completely blocked by cycloheximide and actinomycin D. In contrast to granule neurons, the treatment with NMDA had no significant effect on the activity of glutaminase and glutamine synthetase in cultured cerebellar astroglial cells. Our present results on glutaminase enzyme would indicate that an increase in the cellular concentration of free Ca²⁺ mediated through the NMDA induced increase in Ca²⁺ conductance, leads to long term changes in differentiating cerebellar granule neurons, and it is possible that this kind of physiological stimulation of granule cells is normally provided in vivo by the presynaptic glutamatergic mossy fibers.     

Sites ofd-[H]aspartate accumulation in mouse cerebellar slices

Slices of mouse cerebellar vermis, cut in the parasagittal plane, were incubated for various times (up to 3 h) in the presence of 1μMd-[³H]aspartate, a non-metabolized substrate for the glutamate/aspartate carrier in brain tissue. Light microscopic autoradiography indicated that in regions away from the cut edges of the slices the amino acid accumulated in glia and granule cells. Relatively few grains were seen over Purkinje, Golgi, stellate and basket cells or over white matter. Grain counts over the granule cell layers in the middle parts of the sliced indicated that after short (15 min) exposures to the labelled substrate, non-granule cell areas (which included glia) contained, on average, slightly more grains than granule cells but wit longer exposures (1.5 and 3 h) the relative grain density over granule cells became much higher, possibly because glial uptake preventsd-[³H]aspartate gaining access to neuronal sites in adequate amounts during short incubations and/or because the longer incubations allow time for retrograde migration of the label from parallel fibre terminals to occur. The demonstration of selective uptake ofd-[³H]aspartate into granule cells contrasts with previous autoradiographic results (possible reasons for which are discussed) and supports the notion thatl-glutamate is the transmitter of granule cells. The results also have a bearing on the importance of the metabolic compartmentation of glutamate in relation to its proposed transmitter role.     

Opposite dopaminergic activity in lateral and median hypothalamic nuclei in relation to the feeding effect ofd-Ser-Leu-Enk-Thr (DSLET)

The Leu-enkephalin analogue D-Ser2-Leu-Enk-Thr6 (DSLET) had been shown to enhance feeding in rats, increase dopaminergic activity in the striatum like other opiate agonists, and particularly to decrease dopaminergic activity in the hypothalamus. In this study, the latter effect was found to be localized in the hypothalamic nuclei involved in the regulation of feeding such as the paraventricular (PVN), ventromedian (VMH), dorsomedian (DMH) nuclei and the lateral hypothalamus (LH). DSLET produced the same decrease in dopaminergic activity in the LH as in the whole hypothalamus. In the median nuclei (PVN and VMH and to a lesser extent in the DMH), an opposite effect was observed, resembling that in the striatum. The relevance of these opposite variations with regard to the feeding effect of DSLET is discussed. The decreased dopaminergic activity in the LH would appear to be the most specifically related to the behavioural effect given the known role of dopamine in this region. These data reconcile apparently contradictory aspects of the role of dopamine and the functional opposition between the lateral and median hypothalamus in food intake control.     

Cytoplasmic calcium elevation in hippocampal granule cell induced by perforant path stimulation andl-glutamate application

Calcium-dependent fluorescence of a Ca²⁺ indicator (fura-2) loaded in the slice of guinea pig hippocampus was measured by a m microscope/video-camera/photometry system. Tetanic stimulation of the perforant path (PP) or application ofl-glutamate caused increment of the fluorescence from the dendritic and somatic layers of the granule cells in the dentate gyrus. Magnitude of the increment depended on the frequency and intensity of the PP-stimulation or on the dose ofl-glutamate. 2-Aminophosphonovaleric acid, a glutamate-receptor antagonist, suppressed both PP-stimulus-induced andl-glutamate-evoked responses, while tetrodotoxin blocked the former only. Thus the fluorescence increment should represent an elevation of Ca²⁺ concentration in the postsynaptic cytoplasm of the granule cells.     

Effect ofd- andl-α-aminoadipate on the efflux ofl-aspartate,l-glutamate and γ-aminobutyrate from superfused rat brain slices

d-α-Aminoadipate (d-AA) andl-α-aminoadipate (l-AA) were found to significantly reduce spontaneous efflux of [¹⁴C]l-aspartate from preloaded rat brain slices. Onlyd-AA significantly reduced spontaneous efflux of [¹⁴C]l-glutamate and [³H]γ-aminobutyric acid (GABA);l-AA reduced but not significantly the efflux of these 2 labeled amino acids.d-AA reduced K⁺-stimulated release of [¹⁴C]l-aspartate and [^14]C]l-glutamate significantly, andl-AA that of [³H]GABA significantly. Since bothd-AA andl-AA inhibit the uptake ofl-aspartate,l-glutamate and GABA, their effects on the efflux of these amino acids are more specific. These results also suggest that it is unlikely that the depressant effect ofd-AA, and the excitant effect ofl-AA on neurons when applied locally by iontophoresis are secondary to the accelerated or decelerated release of more specific transmitter amino acids from neighboring cells.     

Effects ofd-Ala-Met-enkephalinamide microinjections placed into the bed nucleus of the stria terminalis upon affective defense behavior in the cat

This study examined the effects of intracerebral injections ofd-Ala²-Met⁵-enkephalinamide (DAME) upon hypothalamically eliited hissing behavior in the cat. The bed nucleus of the stria terminalis (BNST) was selected for investigation because of its anatomical connections with the medial hypothalamus, its relatively high concentrations of enkephalins and opiate receptors and its demonstrated ability to modulate hypothalamically elicited aggressive reactions in the cat. DAME microinjected into the BNST in 1.0 or 10.0 μg/0.5 μl quantities resulted in significant dose dependent increases in mean latencies for elicitation of the hissing response. Suppression of hissing following the 1.0 μg dose of DAME was selectively diminished by prior administration of naloxone. These findings suggest that the opiate receptors within the BNST play a role in the regulation of the hissing component of hypothalamically elicited affective defense behavior.     

N-methyl-d-aspartate stimulates dopamine release through nitric oxide formation in the nucleus accumbens of rats

Intracerebral microdialysis technique was utilized to study the effect ofN^G-nitro-l-arginine, a nitric oxide (NO) synthase inhibitor, onN-methyl-d-aspartate (NMDA)-induced dopamine overflow in the nucleus accumbens of unanesthetized, freely moving rats. Perfusion of 1 and 3 mM NMDA through the microdialysis probe dose-dependently increased the extracellular dopamine level in the nucleus accumbens. Coapplication of 0.5 mMd-(−)-2-amino-5-phosphonovaleric acid (D-AP5), a selective and competitive NMDA receptor antagonist, significantly reduced the dopamine overflow induced by 3 mM NMDA. Perfusion of 0.5 mMN^G-nitro-l-arginine alone did not affect the basal dopamine level, whereas it suppressed the NMDA-evoked dopamine overflow in the nucleus accumbens when concurrently applied with 3 mM NMDA. These results suggest that NO mediates, at least in part, dopamine release resulting from NMDA receptor activation in the nucleus accumbens of rats.     

Effect of aspartame on N-methyl-d-aspartate-sensitive l-[H]glutamate binding sites in rat brain synaptic membranes

Aspartame (l-aspartyl-l-phenylalanine methyl ester), an artificial low-calorie sweetener, was shown to dose-dependently inhibitl-[³H]glutamate binding to its N-methyl-d-aspartate-specific receptors.l-Aspartic acid, a major endogenous metabolite of aspartame, inhibited the binding more stronger than aspartame, while the other metabolites,l-phenylalanine and methanol, had no effect at the same concentration. Aspartame caused a significant change in the affinities ofl-[³H]glutamate binding without altering the V_max values of the binding, suggesting the inhibition is competitive. These in vitro findings suggested that aspartame may act directly on the N-methyl-d-aspartate-sensitive glutamate recognition sites in the brain synaptic membranes.     

Protection by ethanol of cortical neurons from N-methyl-d-aspartate-induced neurotoxicity is associated with blocking calcium influx

Effect of ethanol on N-emthyl-d-aspartate (NMDA)-induced neurotoxicity in rat dissociated cortical cells (8–12 day cultures) was studied. Treatment of cells with NMDA (50 and 500 μM) for 15 min caused cytotoxic effects on the cells, as examined by microscopic observations and lactate dehydrogenase release from cells 18 h after the treatment. Ca²⁺ is essential for these effects in medium during treatment. Presence of ethanol (50–300 mM) simultaneously with NMDA protected cells from the cytotoxicity depending on the concentration of ethanol. Calcium accumulation in cells on addition of NMDA, as monitored by fluorescence ratio (F₄₀₅/F₄₈₅) of Indo-1-preloaded cortical cells, was also decreased depending on the concentration pf added ethanol. APV (200 μM) and ketamine (100 μM) blocked both the cytotoxicity and cellular calcium accumulation due to NMDA. These results suggest that ethanol effects its protection of neurons from NMDA-induced cytotoxicity by blocking the receptor-mediated calcium influx.