Intranasal administration of plasmid DNA nanoparticles yields successful transfection and expression of a reporter protein in rat brain.

Viral vectors are a commonly used method for gene therapy because of their highly efficient transduction of cells. However, many vectors have a small genetic capacity, and their potential for immunogenicity can limit their usefulness. Moreover, for disorders of the central nervous system (CNS), the need for invasive surgical delivery of viruses to the brain also detracts from their clinical applicability. Here, we show that intranasal delivery of unimolecularly compacted DNA nanoparticles (DNA NPs), which consist of single molecules of plasmid DNA encoding enhanced green fluorescent protein (eGFP) compacted with 10 kDa polyethylene glycol (PEG)-substituted lysine 30-mers (CK30PEG10k), successfully transfect cells in the rat brain. Direct eGFP fluorescence microscopy, eGFP-immunohistochemistry (IHC) and eGFP-ELISA all demonstrated eGFP protein expression 2 days after intranasal delivery. eGFP-positive cells were found throughout the rostral-caudal axis of the brain, most often adjacent to capillary endothelial cells. This localization provides evidence for distribution of the nasally administered DNA NPs via perivascular flow. These results are the first report that intranasal delivery of DNA NPs can bypass the blood-brain barrier and transfect and express the encoded protein in the rat brain, affording a non-invasive approach for gene therapy of CNS disorders.

Intranasal administration of glial-derived neurotrophic factor (GDNF) rapidly and significantly increases whole-brain GDNF level in rats.

Previous studies have shown that glial cell line-derived neurotrophic factor (GDNF) exerts significant neuroprotective effects on substantia nigra (SN) neurons in the rat 6-hydroxydopamine (6-OHDA) model of Parkinson's disease (PD). In this study we used enzyme-linked immunosorbent assay (ELISA) to determine GDNF brain levels and distribution to target regions (i.e. striatum and SN) following intranasal administration of GDNF at different time points after administration. Brain levels increased significantly within 1h following a single 50-µg dose of GDNF in a liposomal formulation, returning to baseline by 24h. In a second study, different doses of GDNF (10-150 µg) in phosphate-buffered saline (PBS) were studied at the 1-h time point. Dose-dependent increases in brain GDNF levels were observed with apparent saturation of uptake at doses above 100 µg. Liposomes delivered 10-fold more GDNF to brain than PBS despite yielding similar neuroprotective efficacy in the 6-OHDA model, suggesting incomplete release of GDNF from liposomes in tissue. In a third study, autoradiography was performed on brain sections taken 1h after intranasal (125)I-labeled GDNF. Radioactivity was detected throughout the brain along the rostral-to-caudal axis, indicating that nasally administered GDNF can reach target areas. Collectively, these results demonstrate that intranasal administration of GDNF in liposomes or PBS achieves significant increases in GDNF in target brain areas, supporting use of intranasal administration as a non-invasive means of delivering GDNF to the brain to protect dopamine neurons and arrest disease progression in PD.

Diazepam potentiates GABA-, but not adenosine-mediated, inhibition of neurons of the nigral pars reticulata.

Experiments were conducted to assess the relative roles of gamma-aminobutyric acid (GABA) and adenosine in mediating the inhibition of neuronal activity by diazepam injected intravenously. Recent studies have shown that benzodiazepines inhibit, in a dose-dependent manner, the firing of neurons in the substantia nigra pars reticulata. In support of a predominantly GABAergic mechanism for this inhibitory action, a small dose of diazepam (50 micrograms/kg, i.v.), which itself had little effect on cell firing, significantly potentiated the inhibitory responses of neurons of the pars reticulata to muscimol, a potent GABA agonist given intravenously, and significantly and selectively potentiated the inhibition of reticulata neurons by GABA applied iontophoretically. In contrast to their extreme sensitivity to GABAergic inhibition, neurons of the pars reticulata were comparatively insensitive to systemically and iontophoretically administered adenosine-related drugs. However, in those instances when inhibitions could be achieved with iontophoretically applied adenosine-5'-monophosphate, the inhibitory responses were not significantly modified by a 50 micrograms/kg (i.v.) dose of diazepam. These findings, considered in light of differences in GABA and adenosine receptor densities within the substantia nigra, suggest that the benzodiazepine-induced inhibition of neurons of the nigral pars reticulata most likely involves potentiation of GABA but not adenosine-mediated influences.

Efficacy and potency comparisons among aporphine enantiomers: effects on dopamine neurons in substantia nigra of rat.

Extracellular single unit recording studies were carried out on male rats to determine the responses of dopamine neurons of the substantia nigra to intravenous administration of the enantiomers of the aporphine congeners, apomorphine (APO), N-n-propylnorapomorphine (NPA) and 11-hydroxy-N-n-propylnorapomorphine (11-OH-NPa). The R-(-)-configuration was found to be the most critical determinant of the efficacy and potency of the agonists. All R-(-)-aporphines were full agonists, able to inhibit completely firing of dopamine cells. The order of potencies, defined by the ID50s, was: (-)NPA, 2.0 +/- 0.4 nmol/kg greater than (-)11-OH-NPa, 4.7 +/- 0.7 nmol/kg greater than (-)APO, 18.0 +/- 4.0 nmol/kg. Thus, potency was increased about 9-fold by replacing the 6N methyl of APO with an n-propyl (NPA). Conversely, the 10-hydroxy was not essential for agonist activity (11-OH-NPa) but could increase potency. In the S-(+)-series responses varied. (+)N-n-Propylnorapomorphine exhibited agonist properties and could fully inhibit dopamine cells, but its potency was low (ID50 1550 nmol/kg); (+)APO produced only slight but significant decreases in firing at large (8434 nmol/kg) doses and (+)11-OH-NPa was devoid of efficacy in that it caused no significant changes in firing. Despite their loss of efficacy and potency, the (+)-enantiomers apparently did retain affinity for DA receptors, since they could act as antagonists if given before (-)APO or NPA. These findings suggest that stereochemical conformation and key structural elements of the aporphines are interactive in determining agonist efficacy and potency within this physiological test system.

Differential effects of D1 and D2 dopamine receptor agonists on substantia nigra pars reticulata neurons.

Dopamine was shown in previous studies to exert a dual effect on non-dopaminergic neurons of the substantia nigra pars reticulata: it increases the firing rates of about 50% of cells, and consistently lessens the ability of iontophoretically applied or endogenously released GABA to inhibit their firing. These studies were undertaken to determine (1) whether the two effects could occur independently and, (2) whether different dopamine receptor subtypes might mediate the two responses. Extracellular, single unit activities of pars reticulata neurons were monitored in male rats anesthetized with chloral hydrate. Repeated 30-s iontophoretic pulses of GABA were delivered at an ejection current sufficient to inhibit cell firing by at least 50%, but not totally. After establishing a consistent response to GABA, co-iontophoresis of a test compound was initiated to determine its effects on basal firing rates and responsiveness to GABA. When acetylcholine and glutamate were evaluated in the test paradigm using ejection currents which excited cells by 54.0 +/- 4.9%, neither compound consistently altered the inhibition elicited by GABA. This confirmed that increases in cell firing could occur without concurrent GABA-attenuating effects, and supported the contention that the dual effects of dopamine could be dissociated and perhaps independently mediated. To examine whether the effects of dopamine involve actions at different dopamine receptor subtypes within the nigra, the D1 agonist SKF 38393 and the D2 agonist LY 171555 were substituted in the procedure. Applications of R,S(+/-)-SKF 38393 caused current-dependent increases in firing with a maximal increase at 8 nA of 55 +/- 18% above baseline (n = 14). The excitatory effect appeared to be D1-mediated since R(+)-SKF 38393, but not the inactive S(+)-enantiomer, could elicit the response. Conversely, graded applications of LY 171555 caused only occasional and more modest increases in basal activities, but consistently and markedly attenuated responses to GABA, decreasing GABA's inhibitory potency by 60.9 +/- 4.3% at 10 nA (n = 17). These results provide support for discrete roles of D1 and D2 receptors in substantia nigra pars reticulata, and suggest mechanistically distinct ways by which dendritically released dopamine could act to modify basal ganglia output from this region.

Inhibition of substantia nigra dopamine cell firing by R(-)-N-n-propylnorapomorphine: electrophysiological and autoradiographic studies after regional inactivation of dopamine receptors by microinjection of N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline.

The irreversible receptor inactivator, N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ), was injected into rat striatum or substantia nigra to study potential contributions of dopamine receptors in each area to the inhibition of substantia nigra (A9) dopamine cell firing by i.v. R(-)-N-n-propylnorapomorphine (NPA), a dopamine agonist. Extracellular, single unit recording studies showed that the numbers of active dopamine cells, basal firing rates and responses to i.v. R(-)-NPA were unchanged a day after striatal EEDQ injections, despite significant losses of striatal D1 and D2 receptors (confirmed by autoradiography). These results indicate that striatal receptors do not control the basal activity of A9 neurons, nor do they mediate inhibitions of firing by R(-)-NPA. Microinjections of EEDQ into substantia nigra, however, inactivated 75-78% of nigral D1 and D2 receptors and reduced the number of active dopamine cells and slightly increased firing rates. Moreover, dose-response curves to R(-)-NPA were shifted 10-fold to the right and the maximum inhibitory response was depressed. Furchgott analysis of the dose-response curves yielded a steep occupancy-response curve with maximum (> 95%) inhibition of firing at only 24% receptor occupation (i.e., 76% reserve). Thus, the substantial (approximately 70%) receptor reserve previously shown to exist for inhibition of dopamine cell firing by i.v. R(-)-NPA20,21 appears to be intrinsic to the nigra. To assess contributions of nigral D1 and D2 receptors to this response, selective inactivation of each receptor subtype was achieved (confirmed autoradiographically) by treating rats with SCH 23390 (4 mg/kg) or S(-)eticlopride (2 mg/kg), respectively, 30 min before intranigral EEDQ. Selective D2, but not D1, receptor inactivation produced rightward shifts and depressed the maximum of the R(-)-NPA dose-response curve in a manner like that observed after non-selective inactivation of nigral dopamine receptors. Unexpectedly, pretreatment with SCH 23390 (to protect D1 receptors) also produced a modest rightward shift in the R(-)-NPA dose-response curve, suggesting a slight role for D1 receptors in this response. These results indicate that inhibition of A9 dopamine cell firing by i.v. R(-)-NPA is mediated by dopamine receptors located in substantia nigra, but not striatum and confirm the predominant role of nigral D2 receptors.

Irreversible receptor inactivation reveals differences in dopamine receptor reserve between A9 and A10 dopamine systems: an electrophysiological analysis.

Partial receptor inactivation was used as a tool to examine whether differences in receptor reserve exist between the dopamine receptor populations which mediate responses of substantia nigra (A9) and ventral tegmental area (A10) dopamine neurons to dopamine agonist drugs. The irreversible receptor inactivator, N-ethoxycarbonyl-2-ethoxy-1,2- dihydroquinoline (EEDQ), was administered to rats intraperitoneally at a dose of 6 mg/kg (in an ethanol-water vehicle). Approximately 24 h after EEDQ treatments, extracellular, single-unit recording experiments were carried out. In the first series of experiments, dose-response curves were constructed for the inhibition of A9 and A10 dopamine cell firing by intravenous administration of the potent dopamine agonist, R-(-)-N-n-propylnorapomorphine (NPA). For the A9 dopamine cell group, EEDQ pretreatments caused a 3-fold rightward shift in the NPA dose-response curve (ED50S, 0.3 vs 0.8 micrograms/kg for vehicle- and EEDQ-treated rats, respectively), but there was no change in the maximum attainable response (greater than 95% inhibition of cell firing). For A10 neurons, the same EEDQ treatments produced a greater rightward shift in the dose-response curve to NPA (ED50s, 0.6 vs 5.4 micrograms/kg for vehicle- and EEDQ-treated rats), and also depressed the maximum response by about 25% relative to the control (vehicle) curve. The dose-response curves from each region were subjected to Furchgott analysis to determine relative receptor occupancy-response relationships for NPA. For the A9 system, a steep, hyperbolic occupancy-response plot revealed that a 50% inhibitory response required only 4% receptor occupancy, while complete (greater than 95%) inhibition of cell firing required about 30% occupancy. This suggests about a 70% receptor reserve for this agonist in inhibiting A9 dopamine cell firing. The occupancy-response curve for A10 cells was less steep with 50% and maximal (greater than 95%) responses occurring when 11 and 70% of receptors were occupied by the agonist, indicating only about a 30% reserve for A10 cell responses to NPA. While the level of 'spare' receptors differed substantially between the two areas, calculated pseudo-KA values were similar (7.7 micrograms/kg for A9 cells and 5.5 micrograms/kg for A10 cells), suggesting no regional differences in receptor affinity. To explore where the differences in receptor reserve might reside, a second series of studies evaluated the effects of iontophoretically applied dopamine and NPA on both cell groups in vehicle- and EEDQ-treated rats.(ABSTRACT TRUNCATED AT 400 WORDS)

Dopamine D2, receptor-mediated modulation of the GABAergic inhibition of substantia nigra pars reticulata neurons.

Neurons of the substantia nigra pars reticulata can be readily and fully inhibited by endogenously released or iontophoretically applied GABA. We have previously shown that co-application of dopamine or the D2-like agonist quinpirole causes a current-dependent attenuation of the inhibitory response of these neurons to GABA. To determine if the modulation of GABA responsiveness was mediated by activation of D2 receptors, effects of iontophoretic quinpirole were examined after various treatments which block or inactivate D2 receptors, or uncouple D2 receptors from their G-proteins. Results showed that the GABA-attenuating effect of quinpirole could be attributed to stimulation of D2 receptors, and not a non-specific effect of the drug, since (1) co-iontophoresis of the D2 antagonist YM 09151-2 antagonized the GABA-modulatory effect of quinpirole, (2) prior intranigral injection of the receptor inactivator N-ethoxy-carbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ; 50 nmol/0.5 ml one day before recording) prevented the response to quinpirole, and (3) prior intranigral injection of the Gi-Go-protein inactivator pertussis toxin (1 mg/ml 0.9% NaCl 24 h before recording) completely abolished the ability of quinpirole to lessen the inhibitory response to GABA. The location of the involved D2 receptors was examined using selective lesioning approaches. Kainic acid lesions of the striatonigral pathway did not prevent the ability of quinpirole to attenuate responses of pars reticulata neurons to GABA. Similarly, in previous studies [59], 6-hydroxydopamine lesions of the adjacent pars compacta dopamine neurons were found not to abolish the GABA-attenuating effect of dopamine. Thus, it appears that the receptors mediating the response are not localized to either striatonigral terminals nor to the adjacent dopamine neurons, leaving open the possibility that the response is mediated by D2 receptors located on pars reticulata neurons. Collectively these results suggest that dendritically released dopamine may act via nigral D2 receptors, perhaps located on pars reticulata neurons themselves, to regulate basal ganglia output from the substantia nigra.

Effects of muscimol and picrotoxin on single unit activity of substantia nigra neurons.

Intravenous administration of the GABA agonist, muscimol, caused dose-dependent increases in the unit activity of substantia nigra pars compacta (dopamine) neurons and an inhibition of nigral pars reticulata cells. The depressant effects of the drug upon reticulata neurons were reversible by subsequent administration of the GABA antagonists, picrotoxin and bicuculline HCl. However, the stimulatory effects of i.v. muscimol upon dopamine neurons were not abolished by these agents. Intravenous administration of picrotoxin alone caused only moderate increases in the activity of dopamine neurons (31% over baseline at 7.0 mg/kg), but markedly stimulated the firing of pars reticulata cells (154% over baseline at 7.0 mg/kg). In spite of the stimulation of dopamine neurons after i.v. muscimol, microiontophoresis of GABA and muscimol could inhibit the firing of both pars compacta and pars reticulata cells, although the reticulata neurons were much more sensitive to the inhibitory actions of these agents than the dopamine neurons. Considered together, these studies suggest that a population of neurons in the substantia nigra pars reticulata have the capacity to be more affected by a major GABA input to the nigra than the pars compacta dopamine neurons. The results further suggest that if the dopamine cells are regulated by GABAergic neurons of the striatonigral pathway, their regulation must be indirect and could involve a second inhibitory neuron within the nigra.

Kindling does not cause persistent changes in firing rates or transmitter sensitivities of substantia nigra pars reticulata neurons.

These studies were undertaken to determine whether the kindling process induces persistent alterations in the functional status of neurons of the substantia nigra pars reticulata, a brain area identified previously as a site important in regulating the expression of generalized motor seizures. Extracellular, single-unit recordings of pars reticulata neurons were made in chloral hydrate-anesthetized, fully kindled rats (2-3 weeks after the last seizure), or unkindled control rats of the same age and weight. Kindling caused no alterations in several electrophysiological parameters examined. For instance, neither the number of active pars reticulata cells encountered, nor their firing rates, were significantly different between kindled and control groups. In addition, kindling failed to alter the sensitivities of pars reticulata neurons to iontophoretic application of two inhibitory transmitters, gamma-aminobutyric acid and glycine, and two transmitters that excite these cells, glutamate and acetylcholine. These results suggest that while kindling produces enduring increases in seizure susceptibility, it causes no persistent interictal changes in either basal activity or several measures of transmitter sensitivity of substantia nigra pars reticulata neurons.

Electrophysiological and behavioral output of the rat basal ganglia after intrastriatal infusion of d-amphetamine: lack of support for the basal ganglia model.

Dopamine, by acting upon D1 and D2 dopamine receptors located on striatonigral and striatopallidal neurons, respectively, has been postulated to inhibit output from the substantia nigra pars reticulata (SNpr) and internal pallidal segment (GPi). The inhibition of the SNpr/GPi should, in turn, disinhibit the thalamus to facilitate movement. The present study tests this prediction in intact (unlesioned) rats by attempting to correlate changes in the single unit activities of SNpr neurons with motor (i.e. behavioral) responses in the 20-30 min after infusions of d-amphetamine into the striatum. Unilateral injections of amphetamine (20 microg/microl) into either the dorsal-rostral, central, or ventral-lateral striatum failed to appreciably alter behavior and, in parallel electrophysiological studies, failed to consistently or significantly alter the activities of SNpr neurons in either chloral hydrate-anesthetized rats or awake locally anesthetized rats. However, when amphetamine was infused bilaterally into the ventral-lateral striatum (VLS; 20 microg/microl per side), a robust behavioral activation ensued (increased locomotor activity, oral movements, and sniffing) with an onset ranging from immediate to 20 min post-infusion and persisting for at least 40 min. In parallel studies, bilateral amphetamine infusions into VLS also caused changes in the firing frequency of a majority of SNpr neurons. However, the changes in firing were extremely variable and, contrary to expectation, the net population response of SNpr neurons was an increase in firing which corresponded in time with the period of peak behavioral activation. These results show that (i) bilateral but not unilateral activation of striatal dopamine receptors is needed to elicit behavioral and electrophysiological output from the basal ganglia, and (ii) motor activation is apparently not signaled by a generalized inhibition of SNpr firing, as is predicted by the basal ganglia model.

Single unit responses of substantia nigra pars reticulata neurons to apomorphine: effects of striatal lesions and anesthesia.

Many of the behavioral consequences of dopamine system activation are thought to be mediated by substantia nigra pars reticulata output pathways. Extracellular, single unit recording studies were conducted to determine how i.v. administration of the dopamine agonist, apomorphine, affects the activity of these pars reticulata neurons. Results revealed that a 320 micrograms/kg dose of the drug, considered sufficient to stimulate striatal postsynaptic dopamine receptors, caused highly variable changes in reticulata cell firing. Cells exhibited increases, decreases, or no changes in firing. Many cells also displayed marked minute to minute changes in firing. This non-uniform pattern of responses was not related to state of consciousness since similar responses were observed in both chloral hydrate-anesthetized as well as conscious, paralyzed rats. Both the increases and decreases could be reversed by subsequent administration of haloperidol. The variable responses to apomorphine were reduced but not totally prevented by striatal kainic acid lesions, suggesting that changes in striatonigral transmission may account for some but not all of the firing changes which were observed. A lower dose of apomorphine (20 micrograms/kg), thought to act primarily at dopamine cell autoreceptors, had little effect on reticulata cell firing and did not modify the variable responses normally observed after the higher dose. These results contrast strikingly with the consistent excitatory responses to apomorphine which have previously been observed in the globus pallidus and suggest that complex or multiple indirect effects of the drug may contribute to the varied reticulata responses.

Autoradiography of dopamine D2 receptors using [3H]YM-09151-2.

Rat brain dopamine D2 receptors were labeled autoradiographically using the potent and selective benzamide, [3H]YM-09151-2. Saturation binding data, quantified from autoradiograms, showed specific binding of [3H]YM-09151-2 to striatal D2 receptors with a KD of 82 pM and Bmax of 0.696 pmol/mg protein. Binding equilibrium occurred within 3 h, and a dissociation constant of 15 pM was obtained in kinetic studies. Antagonist competition curves were monophasic and displayed an order of potency expected for D2 receptors: (+)-butaclamol greater than (-)-sulpiride greater than SCH 23390 greater than mianserin. Competition by dopamine resulted in a shallow, biphasic displacement curve. The distribution of [3H]YM-09151-2 binding sites matched the known pattern for D2 receptors, with dense labeling in striatum, olfactory tubercles and nucleus accumbens, and moderate levels in substantia nigra pars compacta and mamillary nuclei. Much lower levels of non-specific binding were observed than are typically obtained when using [3H]spiperone as the ligand. The coincident high affinity and selectivity of [3H]YM-09151-2 for D2 sites should make this compound a preferred choice for tritium-based D2 autoradiography.

A physiological role for dopamine as modulator of GABA effects in substantia nigra: supersensitivity in 6-hydroxydopamine-lesioned rats.

Dopamine has been reported to attenuate the inhibitory effects of gamma-aminobutyric acid (GABA) on substantia nigra pars reticulata neurons. Five to six weeks after rats received 6-hydroxydopamine lesions of the nigral dopamine neurons, the ability of iontophoretically applied dopamine to attenuate pars reticulata responses to GABA was increased by 40% (P less than 0.05) with respect to that of unlesioned rats. These results suggest that the dopamine sites which mediate this modulatory interaction may become supersensitive in animals with dopamine-deficient nigras.

Intravenous GABA agonist administration stimulates firing of A10 dopaminergic neurons.

Increasing intravenous doses of two gamma-aminobutyric acid (GABA) agonists, muscimol and 4,5,6,7-tetrahydroisoxazolo-[5,4-c]-pyridin-3-ol (THIP), caused dose-related increases in extracellular, single unit activity of A10 dopaminergic neurons of the ventral tegmental area. Muscimol, the more potent of the two compounds, stimulated firing at doses approximately 15-20 times lower than the doses of THIP required to elicit equivalent excitatory effects. The maximum stimulation achieved was approximately 150 and 140% of the baseline firing rate for muscimol and THIP, respectively.

Suppression of methionine sulfoximine seizures by intranigral gamma-vinyl GABA injection.

Studies were undertaken to determine whether enhancement of GABAergic transmission within the substantia nigra could inhibit seizures caused by the novel, long latency convulsant, L-methionine, sulfoximine (MSO; 200 mg/kg i.p.). Bilateral injections of gamma-vinyl GABA (10 micrograms/side) into several brain sites resulted in varying degrees of protection against MSO-induced seizures. However, significant protection was afforded only when gamma-vinyl GABA was infused into the nigra. The protective effect was reduced when injections were made at sites 2 mm dorsal to the nigra, and was further diminished when the drug was injected into the striatum. These results further support the hypothesis that elevation of cerebral GABA levels is protective against a range of experimentally induced seizures, particularly when the substantia nigra is the target of such manipulations.

GABAergic actions of THIP in vivo and vitro: a comparison with muscimol and GABA.

GABAmimetic actions of 4,5,6,7-tetrahydroisoxazolo-[5,4-c]-pyridin-3-ol (THIP) were evaluated both in vivo and in vitro and compared with the actions of muscimol and GABA. The GABAergic potencies of these agents were assessed in vivo by measuring their ability to inhibit firing of rat substantia nigra pars reticulata neurons after systemic administration and iontophoretic applications, and in vitro by measuring their ability to inhibit 3H-GABA binding to rat cerebellar membranes. The effects of THIP were found to be similar to those of muscimol and GABA with regard to inhibition of reticulata cell firing and 3H-GABA binding. The order of potency was muscimol > GABA > THIP. The magnitude of the differences between drug potencies in iontophoretic studies closely paralleled their relative potencies in binding studies, with muscimol approximately 3 times more potent than GABA and 25-40 times more potent than THIP. After systemic (i.v.) administration, however, muscimol was only 3 times more potent than THIP in inhibiting reticulata cell firing, possibly because THIP passes the blood-brain barrier more readily.

The putative dopamine D3 receptor agonist 7-OH-DPAT: lack of mesolimbic selectivity.

7-Hydroxy-N,N-di-n-propyl-2-aminotetralin (7-OH-DPAT), an agonist with relative selectivity for the dopamine D3 receptor, was examined in several electrophysiological assays to determine whether it exhibits preferential effects in the mesolimbic versus nigrostriatal dopamine systems. Extracellular single unit activities of substantia nigra pars compacta (A9) and ventral tegmental area (A10) dopamine neurons, and caudate-putamen and nucleus accumbens neurons, were recorded in male rats anesthetized with chloral hydrate. Intravenous (+/-)-7-OH-DPAT potently and completely inhibited the firing of both A9 and A10 dopamine neurons (ED50's: 3.5 +/- 0.7 micrograms/kg and 3.9 +/- 0.9 micrograms/kg, respectively). The active enantiomer, (+)-7-OH-DPAT, was 2 to 3 times more potent than the racemic drug (ED50's: 1.2 +/- 0.3 micrograms/kg and 1.7 +/- 0.4 micrograms/kg for A9 and A10 cells, respectively). There were no significant differences in potency for either form in inhibiting A9 and A10 dopamine neurons. In other studies, iontophoretically applied (+)-7-OH-DPAT caused current-dependent inhibitions of spontaneously active or glutamate-driven caudate-putamen and nucleus accumbens neurons (I50 values, 6.5 and 7.9 nA, respectively). Again, no difference in potency between cell populations was noted. Finally, in cell-attached patch-clamp recordings from freshly dissociated rat caudate-putamen neurons, an 85 pS K+ channel known to be activated by dopamine and the "D2-like" agonist quinpirole was also observed with (+/-)-7-OH-DPAT (0.2-1 microM) applied in the patch pipette.(ABSTRACT TRUNCATED AT 250 WORDS)

Behavioural, biochemical and electrophysiological studies on the motor depressant and stimulant effects of bromocriptine.

Bromocriptine (BRC) produced a biphasic behavioural effect in mice; an early depressant phase which lasted for about 1 h and a later stimulant phase which lasted from about 1 to 5 h. The stimulation was blocked with SCH23390. Both phases of activity were accompanied by marked striatal DA autoreceptor effects as indicated by reductions in dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) levels and by a reduction in the accumulation of DOPA (after inhibition of nigrostriatal DA nerve firing and DOPA decarboxylase). However, while the autoreceptor effects were still evident during the behavioural stimulant phase, there was a gradual rise in DOPAC and HVA from 1 to 4 h after injection, indicating a gradually increasing DA turnover. We were unable, using a variety of behavioural and biochemical paradigms, to demonstrate any change in DA autoreceptor sensitivity after one dose of BRC. In electrophysiological studies, however, it was found that prior exposure of rats to one dose of BRC rendered them subsensitive to the rate-inhibiting effects of a second dose of BRC, as measured in anaesthetized animals using extracellular single cell recordings of identified DA neurons in the substantia nigra pars compacta. It is concluded firstly, that the stimulant phase of BRC in mice occurs despite continued occupation of the DA autoreceptors by BRC because adequate endogenous DA is available to provide the required D1 receptor stimulation and secondly, that the terminal autoreceptors in the striatum (as assessed in mice using biochemical techniques) may be regulated differently to the somatodendritic autoreceptors (as assessed electrophysiologically in rats).

Effects of benzodiazepines on single unit activity in the substantia nigra pars reticulata.

Intravenous administration of two benzodiazepines, flurazepam and diazepam, had an inhibitory effect on the firing rates of neurons of the substantia nigra pars reticulata, a brain region with an identified GABAergic innervation. Diazepam was more potent than flurazepam. Bicuculline and picrotoxin, two drugs which block GABAergic transmission, and caffeine and theophylline, two methylxanthines which inhibit benzodiazepine binding, all reversed the inhibition produced by diazepam. The action of theophylline was less consistent than that of caffeine. Similarly, Ro 15-1788, an imidazodiazepine which putatively functions as a specific benzodiazepine antagonist, reversed the diazepam-induced inhibition. These findings are consistent with previous reports which suggest that the benzodiazepines may act through a GABAergic mechanism. In a separate group of experiments, caffeine or Ro 15-1788 was administered alone. While caffeine excited all reticulata, generally had little excitatory effect. These results suggest: 1) that cells of the substantia nigra pars reticulata may not receive a substantial, tonic inhibition mediated by an endogenous benzodiazepine-like substance; and 2) that the methylxanthines may increase reticulata cell firing, at least in part, through mechanisms unrelated to the blockade of benzodiazepine receptors.