Characterization of cognitive impairments and neurotransmitter changes in a novel transgenic mouse lacking Slc10a4.

An orphan member of the solute carrier (SLC) family SLC10, SLC10A4 has been found to be enriched in midbrain and brainstem neurons and has been found to co-localize with and to affect dopamine (DA) homeostasis. We generated an SLC10A4 knockout mouse (Slc10a4(Δ/Δ)) using Cre-targeted recombination, and characterized behavioral measures of motor and cognitive function as well as DA and acetylcholine (ACh) levels in midbrain and brainstem. In agreement with previous studies, Slc10a4 mRNA was preferentially expressed in neurons in the brains of wild-type (Slc10a4(+/+)) mice and was enriched in dopaminergic and cholinergic regions. Slc10a4(Δ/Δ) mice had no impairment in motor function or novelty-induced exploratory behaviors but performed significantly worse in measures of spatial memory and cognitive flexibility. Slc10a4(Δ/Δ) mice also did not differ from Slc10a4(+/+) in measures of anxiety. High-performance liquid chromatography (HPLC) measures on tissue punches taken from the dorsal and ventral striatum reveal a decrease in DA content and a corresponding increase in the metabolite 3,4-dihydroxyphenylacetic acid (DOPAC), indicating an increase in DA turnover. Punches taken from the brainstem revealed a decrease in ACh as compared with Slc10a4(+/+) littermates. Together, these data indicate that loss of SLC10A4 protein results in neurotransmitter imbalance and cognitive impairment.

Specific contributions of N-methyl-D-aspartate receptors in the dorsal striatum to cognitive flexibility.

Behavioral flexibility is known to be mediated by corticostriatal systems and to involve several major neurotransmitter signaling pathways. The current study investigated the effects of inactivation of glutamatergic N-methyl-D-aspartate-(NMDA) receptor signaling in the dorsal striatum on behavioral flexibility in mice. NMDA-receptor inactivation was achieved by virus-mediated inactivation of the Grin1 gene, which encodes the essential NR1 subunit of NMDA receptors. To assess behavioral flexibility, we used a water U-maze paradigm in which mice had to shift from an initially acquired rule to a new rule (strategy shifting) or had to reverse an initially learned rule (reversal learning). Inactivation of NMDA-receptors in all neurons of the dorsal striatum did not affect learning of the initial rule or reversal learning, but impaired shifting from one strategy to another. Strategy shifting was also compromised when NMDA-receptors were inactivated only in dynorphin-expressing neurons in the dorsal striatum, which represent the direct pathway. These data suggest that NMDA-receptor-mediated synaptic plasticity in the dorsal striatum contributes to strategy shifting and that striatal projection neurons of the direct pathway are particularly relevant for this process.

Heparin-binding correlates with increased efficiency of AAV1- and AAV6-mediated transduction of striated muscle, but negatively impacts CNS transduction.

Gene delivery vectors derived from adeno-associated virus (AAV) have great potential as therapeutic agents. rAAV1 and rAAV6, efficiently target striated muscle, but the mechanisms that determine their tropism remain unclear. It is known that AAV6, but not AAV1, interacts with heparin-sulfate proteoglycans (HSPG). HSPGs are not primary receptors for AAV6, but heparin interactions may affect tissue tropism and transduction. To investigate these possibilities, we generated rAAV1 and rAAV6 capsids that do or do not bind heparin. We evaluated the transduction profile of these vectors in vivo across multiple routes of administration, and found that heparin-binding capability influences tissue transduction in striated muscle and neuronal tissues. Heparin-binding capsids transduce striated muscle more efficiently than non-binding capsids, via both intramuscular and intravenous injection. However, rAAV6 achieved greater muscle transduction than the heparin-binding rAAV1 variant, suggesting that there are additional factors that influence differences in transduction efficiency between AAV1 and AAV6. Interestingly, the opposite trend was found when vectors were delivered via intracranial injection. Non-binding vectors achieved robust and widespread gene expression, whereas transduction via heparin-binding serotypes was substantially reduced. These data indicate that heparin-binding capability is an important determinant of transduction that should be considered in the design of rAAV-mediated gene therapies.

Regulation of NKB pathways and their roles in the control of Kiss1 neurons in the arcuate nucleus of the male mouse.

Kisspeptin (Kiss1) and neurokinin B (NKB) (encoded by the Kiss1 and Tac2 genes, respectively) are indispensable for reproduction. In the female of many species, Kiss1 neurons in the arcuate nucleus (ARC) coexpress dynorphin A and NKB. Such cells have been termed Kiss1/NKB/Dynorphin (KNDy) neurons, which are thought to mediate the negative feedback regulation of GnRH/LH secretion by 17ß-estradiol. However, we have less knowledge about the molecular physiology and regulation of Kiss1/Kiss1-expressing neurons in the ARC of the male. Our work focused on the adult male mouse, where we sought evidence for coexpression of these neuropeptides in cells in the ARC, assessed the role of Kiss1 neurons in negative feedback regulation of GnRH/LH secretion by testosterone (T), and investigated the action of NKB on KNDy and GnRH neurons. Results showed that 1) the mRNA encoding Kiss1, NKB, and dynorphin are coexpressed in neurons located in the ARC; 2) Kiss1 and dynorphin A mRNA are regulated by T through estrogen and androgen receptor-dependent pathways; 3) senktide, an agonist for the NKB receptor (neurokinin 3 receptor, encoded by Tacr3), stimulates gonadotropin secretion; 4) KNDy neurons express Tacr3, whereas GnRH neurons do not; and 5) senktide activates KNDy neurons but has no discernable effect on GnRH neurons. These observations corroborate the putative role for KNDy neurons in mediating the negative feedback effects of T on GnRH/LH secretion and provide evidence that NKB released from KNDy neurons is part of an auto-feedback loop that generates the pulsatile secretion of Kiss1 and GnRH in the male.

Dopamine signaling as a neural correlate of consciousness.

The neural correlates of consciousness are largely unknown but many neural circuits are likely to be involved. Our experiments with mice that cannot synthesize dopamine suggest that dopamine signaling is a critical component necessary for the expression of consciousness. Although dopamine-deficient mice are awake and respond to many stimuli, they are unmotivated and have profound deficits in all but the simplest learning tasks. Dopamine-deficient mice are unable to attend to salient sensory information, integrate it with prior experience, store it in long-term memory, or choose appropriate actions. While clearly conscious from a general anesthetic point of view, dopamine-deficient mice have marginal arousal and appear to be virtually unconscious from a behavioral point of view. Restoration of dopamine signaling within the striatum by viral gene therapy strategies restores most behaviors. Therefore, I propose that dopaminergic modulation of glutamatergic inputs from the cortex and thalamus onto medium spiny neurons in the striatum contributes to cognition and the expression of consciousness.

Behavioral and sleep/wake characteristics of mice lacking norepinephrine and hypocretin.

We investigated the interaction between norepinephrine (NE) and orexin/hypocretin (Hcrt) in the control of sleep behavior and narcoleptic symptoms by creating mice that were deficient in both neurotransmitters. Mice with a targeted disruption of the dopamine beta-hydroxylase (Dbh) gene (deficient in NE and epinephrine) or the Hcrt gene were bred to generate double knockouts (DKOs), each single KO (Dbh-KO and Hcrt-KO), and control mice. The duration of wake, non-rapid eye movement (NREM) and REM sleep were monitored by electroencephalogram (EEG)/electromyogram (EMG) recording over a 24-h period, and the occurrence of behavioral arrests was monitored by video/EEG recording for 4 h. Overall, there was very little interaction between the two genes; for most parameters that were measured, the DKO mice resembled either Dbh-KO or Hcrt-KO mice. REM sleep was increased in both DKO and Hcrt-KO mice at night relative to the other groups, but DKO mice had significantly more REM sleep during the day than the other three groups. Sleep latency in response to saline or amphetamine injections was reduced in Dbh-KO and DKO mice relative to other groups. Behavioral arrests, that are frequent in Hcrt-KO mice, were not exacerbated in DKO mice.

Reversal of supersensitive striatal dopamine D1 receptor signaling and extracellular signal-regulated kinase activity in dopamine-deficient mice.

Lesions of dopaminergic nigrostriatal neurons cause supersensitivity to dopamine in the striatum. Previous work has shown that such supersensitivity, an important aspect of rodent models of Parkinson's disease, is associated with anatomically abnormal patterns in the activation of extracellular signal-regulated kinase. After lesions of dopaminergic neurons, dopamine D1-receptor agonists activate extracellular signal-regulated kinase in the dorsal striatum, something not observed in intact animals. Here we used a more selective method of dopamine depletion. Dopamine-deficient mice, in which the tyrosine hydroxylase gene is specifically inactivated in dopaminergic neurons, were used to investigate dopamine D1-receptor-mediated activation of extracellular signal-regulated kinase. In wild-type mice, acute treatment with a dopamine D1-receptor agonist results in activation of extracellular signal-regulated kinase in the nucleus accumbens without activation in the dorsal striatum. In contrast, in dopamine-deficient mice, dopamine D1-receptor-agonist treatment results in activation of extracellular signal-regulated kinase not only in the nucleus accumbens, but also throughout most of the dorsal striatum. Chronic replacement of dopamine by repeated injection of L-DOPA for 36 h reverses this supersensitive extracellular signal-regulated kinase activation. This reversal displays a dorsal to ventral progression such that, by 36 h, extracellular signal-regulated kinase activation is virtually restricted to the nucleus accumbens, as in wild-type mice. The reversal of dopamine D1-receptor activation of extracellular signal-regulated kinase in dopamine-deficient mice following chronic L-DOPA treatment shows that the lack of dopamine, rather than absence of other factors secreted from dopaminergic neurons, is responsible for dopamine supersensitivity.

The anticonvulsant and proconvulsant effects of alpha2-adrenoreceptor agonists are mediated by distinct populations of alpha2A-adrenoreceptors.

The alpha2-adrenoreceptor (AR) is the most investigated noradrenergic receptor with regard to modulation of seizure activity. However, because of the complexity of multiple alpha2-AR subtypes and their distribution, the exact role of this receptor in modulating seizure activity is not clear. alpha2A- and alpha2C-ARs function as both autoreceptors (presynaptic) on noradrenergic neurons, where they regulate norepinephrine (NE) release, and as postsynaptic receptors on neurons that receive noradrenergic innervation, where they regulate the release of other neurotransmitters (heteroreceptor). The nonselective alpha2-AR agonist clonidine produced a proconvulsant effect on seizure susceptibility, while the selective alpha2A-AR agonist guanfacine was anticonvulsant. The effects of both alpha2-AR agonists were absent in alpha2a knockout mice, suggesting that the alpha2A-AR mediates the proconvulsant and anticonvulsant effect of alpha2-AR agonists on seizure susceptibility. To determine whether the alpha2-AR agonists were acting on inhibitory presynaptic autoreceptors to decrease NE release or on postsynaptic receptors on NE target neurons, the effects of clonidine and guanfacine were determined in dopamine beta-hydroxylase knockout (Dbh -/-) mice that lack NE. The anticonvulsant effect of guanfacine persisted in Dbh -/- mice, suggesting that guanfacine may act preferentially on alpha2A-postsynaptic receptors that regulate the action of NE on target neurons. In contrast, the proconvulsant effect of clonidine was lost in Dbh -/- mice, suggesting that clonidine may act on presynaptic autoreceptors to decrease NE release. We hypothesize that the alpha2A-presynaptic autoreceptor is responsible for the proconvulsant effect of alpha2-AR agonists, while the alpha2A-postsynaptic receptor is responsible for the anticonvulsant effect of alpha2-AR agonists. These data help to clarify the inconsistent effects of alpha2-AR agonists on seizure activity.

Role of metallothioneins in peripheral nerve function and regeneration.

The physiological role of the metallothionein (MT) family of proteins during peripheral nerve injury and regeneration was examined in Mt1+ 2 and Mt3 knockout (KO) mice. To this end, the right sciatic nerve was crushed, and the regeneration distance was evaluated by the pinch test 2-7 days postlesion (dpl) and electrophysiologically at 14 dpl. The quality of the regeneration was assessed by light microscopy and immunohistochemical methods. The results show that the regeneration distance was greater in the Mt3 KO than in the Mt1+ 2 KO mice, whereas control mice showed intermediate values. Moreover, the number of regenerating axons in the distal tibial nerve was significantly higher in Mt3KO mice than in the other two strains at 14 dpl. Immunoreactive profiles to protein gene product 9.5 were present in the epidermis and the sweat glands of the plantar skin of the hindpaw of the Mt3 KO group. The improved regeneration observed with the Mt3 KO mice was confirmed by compound nerve action potentials that were recorded from digital nerves at 14 dpl only in this group. We conclude that Mt3 normally inhibits peripheral nerve regeneration.

Peptides that regulate food intake: norepinephrine is not required for reduction of feeding induced by cholecystokinin.

CCK octapeptide (CCK-8) is released by the gut in response to a meal and acts via CCK(A) receptors on vagal afferents to induce satiety. However, the central neural pathways by which peripheral CCK-8 affects feeding are poorly understood. In the present study, we tested the hypothesis that norepinephrine (NE) is necessary for satiety induced by peripheral CCK-8 by using mice lacking dopamine beta-hydroxylase (Dbh(-/-)), the enzyme responsible for synthesizing NE and epinephrine from dopamine. We found that Dbh(-/-) mice are as responsive to the satiating effects of CCK-8 as their normal littermates.

Lack of vesicular zinc in mossy fibers does not affect synaptic excitability of CA3 pyramidal cells in zinc transporter 3 knockout mice.

Zinc is found throughout the CNS in synaptic vesicles of glutamatergic neurons and has been suggested to have a modulatory role in the brain because of its interaction with voltage- and ligand-gated ion channels. We took advantage of zinc transporter 3 knockout mice, which lack vesicular zinc, to study the possible physiological role of this heavy metal in hippocampal mossy fiber neurotransmission. We examined postsynaptic responses evoked by mossy fiber activation, recorded in CA3 pyramidal cells in hippocampal slices prepared from zinc transporter 3 knockout and wild-type mice. Field-potential response threshold and amplitude, input-output curves, and paired-pulse evoked responses were the same in slices from zinc transporter 3 knockout and wild-type mice. Furthermore, neither amplitude nor duration of pharmacologically isolated N-methyl-D-aspartate, non-N-methyl-D-aspartate, GABA(A), and GABA(B) receptor-mediated postsynaptic potentials differed between zinc transporter 3 knockout and wild-type mice. There was no difference in the magnitude of epileptiform discharges evoked by repetitive stimulation or kainic acid application. However, in slices from zinc transporter 3 knockout mice, there was greater attenuation of GABA(A)-mediated inhibitory postsynaptic potentials during tetanic stimulation compared with slices from wild-type animals. We conclude that lack of vesicular zinc in mossy fibers does not significantly affect the mossy fiber-associated synaptic excitability of CA3 pyramidal cells; however, zinc may modulate GABAergic synaptic transmission under conditions of intensive activation.

Genetic comparison of seizure control by norepinephrine and neuropeptide Y.

Epilepsy is a disease of neuronal hyperexcitability, and pharmacological and genetic studies have identified norepinephrine (NE) and neuropeptide Y (NPY) as important endogenous regulators of neuronal excitability. Both transmitters signal through G-protein-coupled receptors, are expressed either together or separately, and are abundant in brain regions implicated in seizure generation. NPY knock-out (NPY KO) and dopamine beta-hydroxylase knock-out (DBH KO) mice that lack NE are susceptible to seizures, and agonists of NE and NPY receptors protect against seizures. To examine the relative contributions of NE and NPY to neuronal excitability, we tested Dbh;Npy double knock-out (DKO) mice for seizure sensitivity. In general, DBH KO mice were much more seizure-sensitive than NPY KO mice and had normal NPY expression, demonstrating that an NPY deficiency did not contribute to the DBH KO seizure phenotype. DKO mice were only slightly more sensitive than DBH KO mice to seizures induced by kainic acid, pentylenetetrazole, or flurothyl, although DKO mice were uniquely prone to handling-induced seizures. NPY contributed to the seizure phenotype of DKO mice at high doses of convulsant agents and advanced stages of seizures. These data suggest that NE is a more potent endogenous anticonvulsant than NPY, and that NPY has the greatest contribution under conditions of extreme neuronal excitability.

Alpha(1) and beta(2) adrenoreceptor agonists inhibit pentylenetetrazole-induced seizures in mice lacking norepinephrine.

It has been known for many years that norepinephrine (NE) is a potent endogenous anticonvulsant, yet there is confusion as to which receptor(s) mediate this effect. This is probably due to multiple factors, including the importance of distinct signaling pathways for different seizure paradigms, a lack of comprehensive pharmacological studies, and difficulty in interpreting existing pharmacological results due to the presence of endogenous NE. We sought to circumvent these problems by testing the anticonvulsant activity of selective agonists for most known adrenoreceptors (ARs) in dopamine beta-hydroxylase knockout (Dbh -/-) mice that lack endogenous NE. Dbh -/- mice are hypersensitive to pentylenetetrazole (PTZ)-induced seizures, demonstrating that endogenous NE inhibits PTZ-induced seizures in the wild type. Pretreatment of Dbh -/- mice with an alpha(1)AR or beta(2)AR, but not an alpha(2)AR or beta(1)AR agonist significantly protected against PTZ-induced seizures. In contrast, only the beta(2)AR agonist showed anticonvulsant activity in heterozygous controls. Furthermore, an alpha(1)AR antagonist exacerbated PTZ-induced seizures in control mice, whereas a beta(2)AR antagonist had no effect. We conclude that activation of the alpha(1)AR is primarily responsible for the anticonvulsant activity of endogenous NE in the murine PTZ model of epilepsy. Endogenous NE probably does not activate the beta(2)AR under these conditions, but exogenous activation of the beta(2)AR produces an anticonvulsant effect.

Induction of stereotypy in dopamine-deficient mice requires striatal D1 receptor activation.

Motor stereotypies are abnormally repetitive behaviors that can develop with excessive dopaminergic stimulation and are features of some neurologic disorders. To investigate the mechanisms required for the induction of stereotypy, we examined the responses of dopamine-deficient (DD) mice to increasing doses of the dopamine precursor L-DOPA. DD mice lack the ability to synthesize dopamine (DA) specifically in dopaminergic neurons yet exhibit robust hyperlocomotion relative to wild-type (WT) mice when treated with L-DOPA, which restores striatal DA tissue content to approximately 10% of WT levels. To further elevate brain DA content in DD mice, we administered the peripheral L-amino acid decarboxylase inhibitor carbidopa along with L-DOPA (C/l-DOPA). When striatal DA levels reached >50% of WT levels, a transition from hyperlocomotion to intense, focused stereotypy was observed that was correlated with an induction of c-fos mRNA in the ventrolateral and central striatum as well as the somatosensory cortex. WT mice were unaffected by C/L-DOPA treatments. A D1, but not a D2, receptor antagonist attenuated both the C/L-DOPA-induced stereotypy and the c-fos induction. Consistent with these results, stereotypy could be induced in DD mice by a D1, but not by a D2, receptor agonist, with neither agonist inducing stereotypy in WT mice. Intrastriatal injection of a D1 receptor antagonist ameliorated the stereotypy and c-fos induction by C/L-DOPA. These results indicate that activation of D1 receptors on a specific population of striatal neurons is required for the induction of stereotypy in DD mice.

Dopamine production in the caudate putamen restores feeding in dopamine-deficient mice.

Dopamine-deficient (DD) mice cannot synthesize dopamine (DA) in dopaminergic neurons due to selective inactivation of the tyrosine hydroxylase gene in those neurons. These mice become hypoactive and hypophagic and die of starvation by 4 weeks of age. We used gene therapy to ascertain where DA replacement in the brain restores feeding and other behaviors in DD mice. Restoration of DA production within the caudate putamen restores feeding on regular chow and nest-building behavior, whereas restoration of DA production in the nucleus accumbens restores exploratory behavior. Replacement of DA to either region restores preference for sucrose or a palatable diet without fully rescuing coordination or initiation of movement. These data suggest that a fundamental difference exists between feeding for sustenance and the ability to prefer rewarding substances.

Leptin-regulated endocannabinoids are involved in maintaining food intake.

Leptin is the primary signal through which the hypothalamus senses nutritional state and modulates food intake and energy balance. Leptin reduces food intake by upregulating anorexigenic (appetite-reducing) neuropeptides, such as alpha-melanocyte-stimulating hormone, and downregulating orexigenic (appetite-stimulating) factors, primarily neuropeptide Y. Genetic defects in anorexigenic signalling, such as mutations in the melanocortin-4 (ref. 5) or leptin receptors, cause obesity. However, alternative orexigenic pathways maintain food intake in mice deficient in neuropeptide Y. CB1 cannabinoid receptors and the endocannabinoids anandamide and 2-arachidonoyl glycerol are present in the hypothalamus, and marijuana and anandamide stimulate food intake. Here we show that following temporary food restriction, CB1 receptor knockout mice eat less than their wild-type littermates, and the CB1 antagonist SR141716A reduces food intake in wild-type but not knockout mice. Furthermore, defective leptin signalling is associated with elevated hypothalamic, but not cerebellar, levels of endocannabinoids in obese db/db and ob/ob mice and Zucker rats. Acute leptin treatment of normal rats and ob/ob mice reduces anandamide and 2-arachidonoyl glycerol in the hypothalamus. These findings indicate that endocannabinoids in the hypothalamus may tonically activate CB1 receptors to maintain food intake and form part of the neural circuitry regulated by leptin.

Removing zinc from synaptic vesicles does not impair spatial learning, memory, or sensorimotor functions in the mouse.

Zinc-enriched (ZEN) neurons are distributed widely throughout the brain and spinal cord. Synaptic vesicle zinc in these neurons is thought to function as a neuromodulator upon its release into the synaptic cleft. Consistent with this possibility, zinc or zinc chelators can alter spatial learning, working memory, and nociception in rodents. Here we use zinc transporter-3 (ZnT3) knockout mice, which are depleted of synaptic vesicle zinc, to assess the consequences of removing this potential neuromodulator on the behavior of adult mice. ZnT3 knockout mice performed equally as well as wild-type mice in the rotarod, pole, and cagetop tests of motor coordination. They exhibited normal thermal nociception in the hot-plate and tail-flick tests, and had similar olfactory, auditory and sensorimotor gating capabilities as wild-type mice. ZnT3 knockout mice behaved similarly as wild-type mice in the open field test and in the elevated plus maze test of anxiety. They exhibited normal learning and memory in the passive avoidance, Morris water maze, and fear conditioning tasks, and normal working and reference memory in a water version of the radial arm maze. We conclude that synaptic vesicle zinc is not essential for mice to be able to perform these tasks, despite the abundance of ZEN neurons in the relevant regions of the CNS. Either the neuromodulatory effects of zinc are not relevant for the tasks tested here, or mice are able to compensate easily for the absence of synaptic vesicle zinc.

A metabolic defect promotes obesity in mice lacking melanocortin-4 receptors.

Melanocortin-4 receptor (Mc4r)-null mice exhibit late-onset obesity. To determine whether aberrant metabolism contributes to the obesity, food consumption by Mc4r-null mice was restricted to (pair-fed to) that consumed by wild-type (WT) mice. Pair-fed Mc4r-null females maintained body weights intermediate to that of WT and nonpair-fed Mc4r-null females, whereas pairfeeding normalized the body weights of Mc4r-null male mice. Fat pad and circulating leptin levels were elevated in both male and female pair-fed Mc4r-null mice compared with WT mice. Oxygen consumption of Mc4r-null mice with similar body weights as WT controls was reduced by 20%. Locomotor activity of young nonobese Mc4r-null males was significantly lower than that of WT males; however, locomotion of young nonobese females was normal. Core body temperature of Mc4r-null mice was normal, and they responded normally to cold exposure. Young nonobese Mc4r-null females were unable to induce uncoupling protein 1 (UCP1) in brown adipose tissue in response to peripheral leptin administration, whereas UCP1 mRNA was increased by 60% in the WT females. These results indicate that Mc4r deficiency enhances caloric efficiency, similar to that seen in the agouti obesity syndrome and in melanocortin-3 receptor-null mice.

Dopamine-deficient mice are hypersensitive to dopamine receptor agonists.

Dopamine-deficient (DA-/-) mice were created by targeted inactivation of the tyrosine hydroxylase gene in dopaminergic neurons. The locomotor activity response of these mutants to dopamine D1 or D2 receptor agonists and l-3,4-dihydroxyphenylalanine (l-DOPA) was 3- to 13-fold greater than the response elicited from wild-type mice. The enhanced sensitivity of DA-/- mice to agonists was independent of changes in steady-state levels of dopamine receptors and the presynaptic dopamine transporter as measured by ligand binding. The acute behavioral response of DA-/- mice to a dopamine D1 receptor agonist was correlated with c-fos induction in the striatum, a brain nucleus that receives dense dopaminergic input. Chronic replacement of dopamine to DA-/- mice by repeated l-DOPA administration over 4 d relieved the hypersensitivity of DA-/- mutants in terms of induction of both locomotion and striatal c-fos expression. The results suggest that the chronic presence of dopaminergic neurotransmission is required to dampen the intracellular signaling response of striatal neurons.