L-myc and N-myc influence lineage determination in the central nervous system.

The N-myc and the L-myc proto-oncogenes are expressed during embryonal development mainly in the developing brain. Studies of their expression in single neuroepithelial cells revealed that neural precursors not yet committed to the glial or the neuronal lineage expressed both genes, but after lineage commitment they expressed either N-myc or L-myc. Moreover, enforced expression of L-myc in the neural precursor cell line 2.3D caused neuronal differentiation, while the expression of N-myc promoted glial differentiation. These results indicate that L-myc and N-myc play critical roles in lineage determination for the central nervous system.

Fetal striatum- and ventral mesencephalon-derived expanded neurospheres rescue dopaminergic neurons in vitro and the nigro-striatal system in vivo.

The pathogenesis of Parkinson's disease (PD) involves ongoing apoptotic loss of dopaminergic neurons in the substantia nigra pars compacta. Local delivery of the trophic factors can rescue dopaminergic neurons and halt the progression of PD. In this study we show that fetal E11 striatum-derived neurospheres and E14.5 ventral mesencephalon (VM) -derived neurospheres (NS E11 and NSvm, respectively) are a source of factors that rescue dopaminergic neurons. First, long-term expanded NS E11 and NSvm rescued primary dopaminergic neurons from serum-deprivation induced apoptosis and promoted survival of dopaminergic neurons for 14 days in vitro and this effect was due to soluble contact-independent factor/s. Second, green fluorescent protein-expressing NS E11 and NSvm grafted into the midbrain of mice with unilateral 6-hydroxydopamine-induced Parkinsonism resulted in partial rescue of the nigro-striatal system and improvement of the hypo-dopaminergic behavioral deficit. Reverse transcription-polymerase chain reaction (RT-PCR) analysis demonstrated that intact NS E11 and NSvm expressed fibroblast growth factor-2, brain-derived neurotrophic factor (BDNF), pleiotrophin, neurotrophin-3, but not glial cell line-derived neurotrophic factor (GDNF). GDNF expression was also undetectable in vivo in grafted NS E11 and NSvm suggesting that NS-derived factor/s other than GDNF mediated the rescue of nigral dopaminergic neurons. Identification of NS-derived soluble factor(s) may lead to development of novel neuroprotective therapies for PD. An unexpected observation of the present study was the detection of the ectopic host-derived tyrosine hydroxylase (TH) -expressing cells in sham-grafted mice and NS E11- and NSvm -grafted mice. We speculate that injury-derived signals (such as inflammatory cytokines that are commonly released during transplantation) induce TH expression in susceptible cells.

Effect of very high dose D-leucine6-gonadotropin-releasing hormone proethylamide on the hypothalamic-pituitary testicular axis in patients with prostatic cancer.

Potent synthetic analogs of gonadotropin-releasing hormone produce parodoxical antireproductive effects when administered chronically. These compounds are minimally toxic and may exhibit no plateau of the dose-response curve even at very high doses. These considerations served as the basis for our systematic evaluation of [D-leucine6-desarginine-glycine-NH2(10)]gonadotropin-releasing hormone (GnRH-A) proethylamide in the very high dose range (i.e., 10-fold larger amounts than previously used). In rats given the analog for 12 wk, prostate, testis, and seminal vesicle weights were suppressed to a greater extent with 200 micrograms q.d. than with 40 micrograms q.d. (P less than 0.01 prostate, less than 0.01 testis, less than 0.01 seminal vesicles), indicating dose-response effects in the very high dose range. 200 micrograms of [D-Leu6-des-Gly-NH2(10]-GnRH-A consistently suppressed leutinizing hormone (LH) values at 6 and 12 wk (basal 71 +/- 9.5; 6 wk 34 +/- 3.8; 12 wk 28 +/- 5 ng/ml) whereas 40 micrograms suppressed LH variably (basal 33 +/- 3.8; 6 wk 17 +/- 3.9; 12 wk 32 +/- 5.2). Testosterone fell to 15 +/- 2.4 and 19 +/- 2.0 ng/100 ml in response to 200 micrograms q.d. and to 27 +/- 6.4 and 22 +/- 7.4 ng/100 ml with the 40-micrograms dose. These findings in the rodent prompted treatment of stage D prostate cancer patients with similarly high doses of [D-Leu6-des-Gly-NH2(10)]-GnRH-A. After treatment for 11 wk with 1,000 or 10,000 micrograms/d of the analog, testosterone and dihydrotestosterone levels transiently rose and then fell into the surgically castrate range (testosterone 19 +/- 4.4 ng/100 ml [D-Leu6-des-Gly-NH2(10)]-GnRH-A vs. surgically castrate 11 +/- 0.9 ng/100 ml, P = NS; dihydrotestosterone 15 +/- 1.7 ng/100 ml GnRH-A vs. surgically castrate 15 +/- 4.1 ng/100 ml. P = NS). However, unlike the chronic stimulatory effect on the pituitary at lower doses, very high dose therapy resulted in profound suppression of plasma and urine LH. Plasma levels fell to the limit of assay detectability, whereas the more sensitive urinary assay detected prepubertal levels of excretion (i.e., 64 +/- 8.4 mlU/h). The highly sensitive rat interstitial cell testosterone bioassay for LH also demonstrated a marked decline in LH to undetectable levels in 17/19 subjects. Clinical results with [D-Leu6-des-Gly-NH2(10)]-GnRH-A simulate those achieved by surgical castration in men with prostatic cancer as suggested by available preliminary data.

Role of the D1A dopamine receptor in the pathogenesis of genetic hypertension.

Since dopamine produced by the kidney is an intrarenal regulator of sodium transport, an abnormality of the dopaminergic system may be important in the pathogenesis of hypertension. In the spontaneously hypertensive rat (SHR), in spite of normal renal production of dopamine and receptor density, there is defective transduction of the D1 receptor signal in renal proximal tubules, resulting in decreased inhibition of sodium transport (Na+/H+ exchanger [NHE] and Na+/K+ATPase activity) by dopamine. To determine if impaired D1 receptor regulation of NHE in proximal tubules is related to hypertension, studies were performed in a F2 generation from female Wistar Kyoto (WKY) and male SHR crosses. A D1 agonist, SKF 81297, inhibited (37.6 +/- 4.7%) NHE activity in brush border membranes of normotensive F2s (systolic blood pressure < 140 mm Hg, n = 7) but not in hypertensive F2s (n = 21). Furthermore, a D1 agonist, SKF 38393, when infused into the renal artery, dose dependently increased sodium excretion in normotensive F2s (n = 3) without altering renal blood flow but was inactive in hypertensive F2s (n = 21). Since the major D1 receptor gene expressed in renal proximal tubules is the D1A subtype, we determined the importance of this gene in the control of blood pressure in mice lacking functional D1A receptors. Systolic blood pressure was greater in homozygous (n = 6) and heterozygous (n = 5) mice compared to normal sex matched litter mate controls (n = 12); moreover, the mice lacking one or both D1A alleles developed diastolic hypertension. The cosegregation with hypertension of an impaired D1 receptor regulation of renal sodium transport and the development of elevated systolic and diastolic pressure in mice lacking one or both D1A alleles suggest a causal relationship of the D1A receptor gene with hypertension.

Targeted disruption of the alpha isoform of the peroxisome proliferator-activated receptor gene in mice results in abolishment of the pleiotropic effects of peroxisome proliferators.

To gain insight into the function of peroxisome proliferator-activated receptor (PPAR) isoforms in rodents, we disrupted the ligand-binding domain of the alpha isoform of mouse PPAR (mPPAR alpha) by homologous recombination. Mice homozygous for the mutation lack expression of mPPAR alpha protein and yet are viable and fertile and exhibit no detectable gross phenotypic defects. Remarkably, these animals do not display the peroxisome proliferator pleiotropic response when challenged with the classical peroxisome proliferators, clofibrate and Wy-14,643. Following exposure to these chemicals, hepatomegaly, peroxisome proliferation, and transcriptional-activation of target genes were not observed. These results clearly demonstrate that mPPAR alpha is the major isoform required for mediating the pleiotropic response resulting from the actions of peroxisome proliferators. mPPAR alpha-deficient animals should prove useful to further investigate the role of this receptor in hepatocarcinogenesis, fatty acid metabolism, and cell cycle regulation.

Genetic interdependence of adenosine and dopamine receptors: evidence from receptor knockout mice.

Dopamine and adenosine receptors are known to share a considerable overlap in their regional distribution, being especially rich in the basal ganglia. Dopamine and adenosine receptors have been demonstrated to exhibit a parallel distribution on certain neuronal populations, and even when not directly co-localized, relationships (both antagonistic and synergistic) have been described. This study was designed to investigate dopaminergic and purinergic systems in mice with ablations of individual dopamine or adenosine receptors. In situ hybridization histochemistry and autoradiography was used to examine the level of mRNA and protein expression of specific receptors and transporters in dopaminergic pathways. Expression of the mRNA encoding the dopamine D2 receptor was elevated in the caudate putamen of D1, D3 and A2A receptor knockout mice; this was mirrored by an increase in D2 receptor protein in D1 and D3 receptor knockout mice, but not in A2A knockout mice. Dopamine D1 receptor binding was decreased in the caudate putamen, nucleus accumbens, olfactory tubercle and ventral pallidum of D2 receptor knockout mice. In substantia nigra pars compacta, dopamine transporter mRNA expression was dramatically decreased in D3 receptor knockout mice, but elevated in A2A receptor knockout mice. All dopamine receptor knockout mice examined exhibited increased A2A receptor binding in the caudate putamen, nucleus accumbens and olfactory tubercle. These data are consistent with the existence of functional interactions between dopaminergic and purinergic systems in these reward and motor-related brain regions.

Bladder wall penetration of intravesical mitomycin C in dogs.

We examined the kinetics of penetration of mitomycin C (MMC) in the dog bladder wall after intravesical instillation of 20 mg/40 ml, a dose used in patients. Bladder tissues were harvested and concentration-depth profiles were established by analysis of thin tissue slices cut parallel to the urothelial surface of the bladder. Tissue concentrations after a dwell time of 5-7 min were similar to those after 30-120 min. In tissues harvested 60 and 75 min after removal of the dose, MMC was not detected in 5 of 6 samples and was less than 1 micrograms/g at the mucosa in the remaining sample, suggesting a rapid "washout" of the drug. The rapid equilibrium between the drug in urine and bladder tissue indicates that the duration of exposure of the bladder wall tissue was approximately equal to the dwell time of intravesical therapy. Tissue concentrations declined log-linearly with respect to the depth of penetration. The concentration immediately underneath the urothelium (C0) showed considerable intra- and interanimal variability. Bladder distention appeared to increase C0 by several fold. C0 ranged from 2 to 275 micrograms/g wet tissue weight, with a median value of 24 micrograms/g, or 11 micrograms/g when two animals with distended bladders were excluded. MMC concentrations in 3 different sites of the same bladder varied up to 5-fold. Within the capillary-perfused mucosa and muscularis (between 50 and 2000 microns from the urothelial surface), concentrations decreased by 50% for each 500-microns distance. The median concentration at 2000 microns was 1 microgram/g (n = 24). At 2000-3000 microns, tissue concentrations in most (18 of 24) specimens either declined to an asymptotic value or were lower than the detection limit of 0.1 microgram/g. Concentrations in the bladder contents were 200-500 micrograms/ml, the average tissue concentration from 50 to 3000 microns was 10 micrograms/g, and plasma concentrations were less than 0.1 microgram/ml. This supports the therapeutic advantage of intravesical therapy of high local drug concentrations while minimizing systemic exposure. A comparison of the urine concentration and C0 indicated a 30-fold decline in concentration across the urothelium. This suggests the importance of the urothelium as a barrier to MMC absorption. A separate study in our laboratories showed that 16 micrograms/ml of MMC was needed to produce a 90% inhibition of the labeling index of explants of human bladder cancers located in the urothelium (Ta tumor, TNM classification), 25 micrograms/ml in the lamina propria (T1 tumors), and 43 micrograms/ml in the muscle layer (T2 tumors).(ABSTRACT TRUNCATED AT 400 WORDS)

Pharmacokinetics of intravesical mitomycin C in superficial bladder cancer patients.

Intravesical mitomycin C (MMC) therapy is used to treat superficial bladder cancer. This study was to establish the intra- and intersubject variabilities in the systemic (plasma) and target site (bladder) exposure to the drug and to identify the factors which contribute to these variabilities. The pharmacokinetics of MMC were studied in 10 patients. Treatment consisted of transurethral tumor resection followed by six weekly intravesical treatments with MMC (20 mg in 40 ml of water). The dosing solution was maintained in the bladder for 2 h. Pharmacokinetic studies were performed at the time of the first, fourth, and sixth or first, second, and fourth treatments with MMC for a total of 28 treatments. Concentration-time profiles of the plasma and bladder contents (i.e., urine), urine volumes, and urine pH were determined during and for up to 4 h after intravesical administration. Maximal plasma MMC concentrations averaged 43 ng/ml (range, 2.1-180.5 ng/ml) in treatment 1. In comparison, the MMC plasma concentration for myelosuppression reported in the literature is 400 ng/ml. Maximal plasma concentrations in treatments 2, 4, and 6 were at least 4-fold lower than those in treatment 1 and in most cases were below the detection limit of 0.5 ng/ml. This indicates that the absorption of MMC during the later treatments was less than in the first treatment given shortly after surgery. Urinary MMC concentrations during instillation declined from 519.4 +/- 34.8 micrograms/ml (mean +/- SD) in the dosing solution to 64.6 +/- 39.4 micrograms/ml 2 h after instillation. Thus, the superficial bladder tissue was exposed to drug concentrations 300- to greater than 34,000-fold higher than the plasma-perfused systemic tissues. Intravesical exposure to MMC, as determined by the area under the urine concentration-time curve, showed large intra- and intersubject variabilities (range, 2,185-40,411 micrograms-min/ml). Pharmacokinetic analysis showed that the bladder exposure to MMC inversely correlated with the residual urine volume at the time of drug administration (P less than 0.001), the urine production rate (P = 0.05), and the rate of drug removal by degradation and absorption during therapy (P less than 0.01). At the end of the 2-h treatment, recovery of MMC from the bladder instillate ranged from 1 to 100% and correlated with the urine pH at the time of removal (P less than 0.001). At pH between 5 and 5.5, less than 30% of the dose was recovered.(ABSTRACT TRUNCATED AT 400 WORDS)

The role of dopamine receptors in regulating the size of axonal arbors.

Factors that regulate terminal arbor size of substantia nigra pars compacta (SNpc) neurons during development and after injury are not well understood. This study examined the role of dopamine receptors in regulating arbor size. Terminal arbors were examined in mice with targeted deletion of the D1 or D2 dopamine receptor [D1(-/-) and D2(-/-) mice, respectively]. Terminal trees were also examined after treatment with receptor blockers and after partial SNpc lesions. Immunohistochemistry was performed, and the number of SNpc neurons and dopaminergic terminals in the striatum was estimated. The number of dopaminergic SNpc neurons were reduced in D1(-/-) and D2(-/-) mice. Density of dopaminergic terminals was unchanged in D1(-/-) mice and increased in D2 (-/-) mice. Steady-state striatal DA and DOPAC levels revealed that dopamine activity was enhanced in D2(-/-) mice but reduced in D1(-/-) mice. Two months after partial SNpc lesions, striatal terminal density was normal in both wild-type and D1(-/-) mice but reduced in D2(-/-) mice. Administration of DA receptor antagonists resulted in larger terminal arbors in D1(-/-) and wild-type mice, whereas D2(-/-) mice showed no change in terminal density. Functional blockade of the D2R during development or in the adult brain results in increased axonal sprouting. Partial SNpc lesions resulted in compensatory sprouting, only in mice with functional D2R. These results suggest that individual dopaminergic axons in D2(-/-) mice have reached maximal arbor size. We conclude that the D2 receptor may play a role in modulating the extent of the terminal arbor of SNpc neurons.

Phase II trial of interferon-beta-serine in metastatic renal cell carcinoma.

Interferon-beta-serine (IFN-beta-ser) is a muteine, recombinant IFN that is tolerated at a dose fivefold to 10-fold higher than IFN-alfa and interacts with the same cell membrane receptor as IFN-alfa. We hypothesized that at high doses IFN-beta-ser might induce a higher response rate than IFN-alfa in metastatic renal cell carcinoma. We undertook a phase II trial of IFN-beta-ser in patients with metastatic renal cell carcinoma. Patients were treated three times each week by a 2-hour intravenous infusion. Doses were escalated weekly (.25 to 5.5 mg, 1 mg = 180,000,000 U) until the maximum-tolerated treatment dose (MTTD) was determined. The MTTD is defined as one dose level less than that which caused grade 3 toxicity and was subsequently administered three times weekly for at least 4 weeks. Twenty-nine patients were entered, and 25 were assessable for response and toxicity. The performance status was 0-1 in all patients and only one patient received previous chemotherapy. The MTTD dose was 2.5 mg (range, 0.5 to 5.5 mg per treatment), although in 10 patients, doses were later deescalated because of cumulative toxicity. Initial dose-limiting toxicity and cumulative toxicity were fatigue, malaise, and fever in most patients. Hepatic transaminitis, neutropenia, and elevation of serum creatinine were also observed but were not dose-limiting. There was one complete response (CR) and four partial responses (PRs). All responses but one occurred in pulmonary metastases. The median time to response was 26 days (range, 17 to 102 days). These data demonstrate that IFN-beta-ser given in high doses exhibits significant antitumor activity in renal cell carcinoma; however, the objective response rate is 20%. This is no higher than previous IFN studies; therefore, we reject the hypothesis than IFN-beta-ser at high doses may induce a greater response rate than IFN-alfa. However, we did observe more responses than were seen in a similar trial undertaken with lower dose IFN-beta serine in renal cell carcinoma.

Projections from the substantia nigra pars reticulata to the motor thalamus of the rat: single axon reconstructions and immunohistochemical study.

This is a study in the rat of the distribution of specific neurotransmitters in neurones projecting from the substantia nigra reticulata (SNR) to the ventrolateral (VL) and ventromedial (VM) thalamic nuclei. Individual axons projecting from the SNR to these thalamic nuclei have also been reconstructed following small injection of the anterograde tracer dextran biotin into the the SNR. Analysis of reconstructions revealed two populations of SNR neurones projecting onto the VL and VM thalamic nuclei. One group projects directly onto the VM and VL, and the other projects to the VM/VL and to the parafascicular nucleus. In another set of experiments Fluoro-Gold was injected into the VL/VM to label SNR projection neurones retrogradely, and immunohistochemistry was performed to determine the distribution of choline acetyltransferase (ChAT), vesicular acetylcholine transporter (VAChT), gamma-aminobutyric acid (GABA), and glutamate in Fluoro-Gold-labelled SNR projection neurones. Most SNR-VL/VM thalamic projection neurones were immunoreactive to acetylcholine or glutamate, whereas only 25% of the projection neurones were found to be immunoreactive to GABA.

Topographical evaluation of the phenotype of spontaneous behaviour in mice with targeted gene deletion of the D1A dopamine receptor: paradoxical elevation of grooming syntax.

The phenotype of spontaneous behaviour in mice with targeted gene deletion of the DIA dopamine receptor was investigated topographically. Via direct visual observation, individual elements of behaviour were resolved and quantified using an ethologically-based, rapid time-sampling behavioural check-list procedure. Relative to wildtypes (D1A+/+), D1A-null (-/-) mice evidenced over initial exploration significant reductions in rearing free, sifting and chewing, but significant increases in locomotion, grooming and intense grooming. Sniffing and rearing to a wall habituated less readily in D1A-null mice such that these behaviours occurred subsequently to significant excess: increases in locomotion were persistent. The ethogram of spontaneous behaviour in D1A-null mice was characterised by neither 'hypoactivity' or 'hyperactivity' but, rather, by prominent topographical shifts between individual elements of behaviour that could not be encapsulated by either term. Given the substantial body of evidence that grooming and particularly intense grooming constitute the most widely accepted behavioural index of D1-like receptor function, the elevation of such behaviour in D1A-null mice was paradoxical; it may reflect (over)compensatory processes subsequent to developmental absence of D1A receptors and/or the involvement of a D1-like receptor other than/additional to the D1A subtype.

Electroencephalographic characterisation of pentylenetetrazole-induced seizures in mice lacking the alpha 4 subunit of the neuronal nicotinic receptor.

Autosomal Dominant Nocturnal Frontal Lobe Epilepsy (ADNFLE) is associated in some kindreds with mutations in the genes encoding the alpha 4 or beta 2 subunits of the neuronal nicotinic acetylcholine receptor (nAChR). Functional characterisation of the described ADNFLE mutations in oocyte preparations has produced conflicting results, with some studies suggesting hypofunction but others showing increased ligand sensitivity or delayed desensitisation. Knockout mice were studied to investigate extreme hypofunction of alpha 4 nAChRs in vivo. Mutant (Mt) and control mice underwent epidural electroencephalographic (EEG) recording for 2 h in the untreated state and for 1 h following administration of the gamma-amino butyric acid (GABA) antagonist, pentylenetetrazole (PTZ, 80 mg/kg). No spontaneous seizures occurred and no EEG differences were observed between the genotypes in drug naïve mice. Following PTZ, however, Mt mice showed markedly increased mortality compared to controls (85 vs 30%, P<0.001). Mts also had a greater number of generalised clonic seizures in the first 40 min following injection. In the same period, the EEGs of Mt mice showed an excess of spikes (P=0.033), multi-spike complexes (P=0.002) and continuous fast activity (P=0.017) compared to controls. These findings demonstrate that intact alpha 4 nAChR subunits provide significant in vivo protection against the proconvulsant effects of GABA antagonism.

Phenotypic resolution of spontaneous and D1-like agonist-induced orofacial movement topographies in congenic dopamine D1A receptor 'knockout' mice.

A novel system was used to assess the role of D(1)-like dopamine receptors in distinct topographies of orofacial movements in mice with congenic D(1A) receptor knockout. Under spontaneous conditions, vertical jaw movements in wild-types declined with time at a rate that was reduced in D(1A) mutants, while horizontal jaw movements emerged progressively in wild-types but not in D(1A) mutants; tongue protrusions were absent in D(1A) mutants, while incisor chattering was initially reduced in D(1A) mutants but rose subsequently to reach the level of wild-types. D(1A) receptors exert a topographically specific role in regulating individual spontaneous orofacial movements, and these involve interactions with psychomotor processes which 'sculpt' behavioural change over time. The anomalous D(1)-like agonist SK&F 83959, which fails to stimulate, and indeed inhibits the stimulation of adenylyl cyclase induced by dopamine, readily stimulated vertical jaw movements, tongue protrusions and incisor chattering, and these response topographies were absent in D(1A) mutants. These results suggest that D(1A) receptors may exert some form of permissive role over orofacial topographies initiated via a novel, putative D(1)-like site not linked to adenylyl cyclase, or that some D(1A) receptors might be coupled to a transduction system other than adenylyl cyclase.

D1 dopamine receptor-deficient mouse: cocaine-induced regulation of immediate-early gene and substance P expression in the striatum.

Psychomotor stimulants such as cocaine alter gene expression in neurons of the striatum. Whereas many of these effects are mediated by D1 dopamine receptors, the involvement of other dopamine receptor subtypes or neurotransmitters is likely. To distinguish between these possibilities, regulation by cocaine of immediate-early genes and genes encoding neuropeptides was analysed in mice that lack functional D1 receptors. Gene expression was examined with in situ hybridization histochemistry. In these animals, cocaine failed to induce the immediate-early genes c-fos and zif 268. In contrast, substance P expression was abnormally increased by this drug. These results demonstrate that some of the effects of cocaine on gene regulation are mediated via D1 receptor-dependent mechanisms, as evidenced by the absence of immediate-early gene induction in D1-deficient mice, whereas others also involve additional, non-D1 receptor mechanisms, as shown for substance P expression.

Late direct and transneuronal effects in mice with targeted expression of a toxin gene to D1 dopamine receptor neurons.

Detailed analysis of a novel transgenic model of basal ganglia disease has been undertaken. In this model the expression of an attenuated form of the diphtheria toxin gene was tightly controlled by D1 dopamine receptor regulatory domains. The behavioral and both direct toxin-mediated and transneuronal effects observed in pups in the first postnatal week have been described. Although younger pups are bradykinetic, older pups have a hyperkinetic syndrome with gait abnormality, postural instability and myoclonic jerks typical of human basal ganglia diseases such as Huntington's disease. As expected, striatal D1 dopamine receptor, dynorphin and substance P transcripts were not detected by in situ hybridization but there was a 27% increase in striatal D2 dopamine receptor messenger RNA and a 65% increase in enkephalin messenger RNA expression. Receptor autoradiographic studies confirmed the lack of D1-class binding in the mutant striatum and in contrast to young pups, a substantial increase in striatal D2-class binding. Autoradiographic quantitation also showed a 30% increase in striatal dopamine transporter binding. In addition to the changes described in the striatopallidal and nigrostriatal pathways, up-regulated dynorphin and substance P messenger RNA expression was also seen in the cortex. The capacity of the developing brain for neurochemical adaptation following injury is dramatic. The results show that primary loss of D1 dopamine receptor-positive striatonigral pathway neurons is sufficient to generate a hyperkinetic phenotype.

Early direct and transneuronal effects in mice with targeted expression of a toxin gene to D1 dopamine receptor neurons.

The neurochemical profile was examined at postnatal day 3-4 in mutant mice generated by in vivo Cre mediated activation of an attenuated diphtheria toxin gene inserted into the D1 dopamine receptor gene locus. An earlier study of this model had shown that D1 dopamine receptor, substance P and dynorphin were not expressed in the striatum. Quantitative in situ hybridization analysis showed an increase in D2 dopamine receptor and enkephalin messenger RNA expression. The nigrostriatal pathway in the mutant pups was intact with a normal number of dopaminergic neurons in the substantia nigra and the ventral tegmental area in addition to a normal pattern of striatal dopamine transporter and tyrosine hydroxylase immunoreactivity. Quantitative analysis of striatal dopamine transporter density using [3H]mazindol showed a reduction of 26% suggesting a degree of transneuronal down-regulation. There was also a 49% reduction of striatal GABA receptor binding and a 36% reduction of striatal muscarinic receptor binding in mutant pups. The number of healthy striatal neuropeptide Y-containing interneurons was also substantially down-regulated in the mutant striatum. In contrast, there was an increase in the number of striatal cholinergic interneurons. Down-regulated cortical GABA receptor and muscarinic receptor binding was also observed in addition to subtle morphological changes in the neuropeptide Y-expressing population of cortical neurons. The changes reflect the early cascade of events which follows the ablation of D1 dopamine receptor-positive cells. Although extensive changes in a number of striatal and cortical neurons were demonstrated, only subtle transneuronal effects were seen in the nigrostriatal pathway.

Conservation of behavioural topography to dopamine D1-like receptor agonists in mutant mice lacking the D1A receptor implicates a D1-like receptor not coupled to adenylyl cyclase.

Though D1-like dopamine receptors [D1A/B] are defined in terms of linkage to the stimulation of adenylyl cyclase, with D1A assumed to be the functionally prepotent subtype, evidence suggests the existence of another, novel D1-like receptor without such coupling. To investigate these issues we challenged mutant mice having targeted gene deletion of the D1A receptor with selective agonists and used an ethologically-based assessment technique to resolve resultant behavioural topography. D1-like-dependent behaviour was substantially conserved in D1A-null mice relative to wild-types following challenge with each of two selective D1-like agents: A 68930 (0.068-2.0 mg/kg s.c.) which exhibits full efficacy to stimulate adenylyl cyclase, and SKF 83959 (0.016-2.0 mg/kg s.c.) which fails to stimulate adenylyl cyclase, and indeed inhibits the stimulation of adenylyl cyclase induced by dopamine. Furthermore, responsivity to the selective D2-like agonist RU 24213 (0.1-12.5 mg/kg s.c.) was conserved in D1A-null mice, indicating the integrity of D1-like:D2-like interactions at the level of behaviour. These data are consistent with behavioural primacy of a D1-like receptor other than D1A [or D1B] that is coupled to a transduction system other than/additional to adenylyl cyclase.

Behavioural assessment of mice lacking D1A dopamine receptors.

Dopamine D1A receptor-deficient mice were assessed in a wide variety of tasks chosen to reflect the diverse roles of this receptor subtype in behavioural regulation. The protocol included examination of exploration and locomotor activity in an open field, a test of sensorimotor orienting, both place and cue learning in the Morris water maze, and assessment of simple associative learning in an olfactory discrimination task. Homozygous mice showed broad-based impairments that were characterized by deficiencies in initiating movement and/or reactivity to external stimuli. Data obtained from flash evoked potentials indicated that these deficits did not reflect gross visual impairments. The partial reduction in D1A receptors in the heterozygous mice did not affect performance in most tasks, although circumscribed deficits in some tasks were observed (e.g., failure to develop a reliable spatial bias in the water maze). These findings extend previous behavioural studies of null mutant mice lacking D1A receptors and provide additional support for the idea that the D1A receptor participates in a wide variety of behavioural functions. The selective impairments of heterozygous mice in a spatial learning task suggest that the hippocampal/cortical dopaminergic system may be uniquely vulnerable to the partial loss of the D1A receptor.