Elizabeth A. Pehek

Pet-1 ETS Gene Plays a Critical Role in 5-HT Neuron Development and Is Required for Normal Anxiety-like and Aggressive Behavior

The central serotonin (5-HT) neurotransmitter system is an important modulator of diverse physiological processes and behaviors; however, the transcriptional mechanisms controlling its development are largely unknown. The Pet-1 ETS factor is a precise marker of developing and adult 5-HT neurons and is expressed shortly before 5-HT appears in the hindbrain. Here we show that in mice lacking Pet-1, the majority of 5-HT neurons fail to differentiate. Remaining ones show deficient expression of genes required for 5-HT synthesis, uptake, and storage. Significantly, defective development of the 5-HT system is followed by heightened anxiety-like and aggressive behavior in adults. These findings indicate that Pet-1 is a critical determinant of 5-HT neuron identity and implicate a Pet-1-dependent program in serotonergic modulation of behavior.

Localization of 5-HT2A receptors on dopamine cells in subnuclei of the midbrain A10 cell group

Considerable evidence suggests that a dysfunction of the dopamine and serotonin (5-hydroxytryptamine or 5-HT) neurotransmitter systems contributes to a diverse range of pathological conditions including schizophrenia, depression and drug abuse. Recent electrophysiological and behavioral studies suggest that 5-HT modulates dopaminergic neurons in the ventral tegmental area via activation of 5-HT(2A) receptors. It is currently unknown if 5-HT(2A) receptors mediate their actions on dopaminergic neurons in the ventral tegmental area via direct or indirect mechanisms. This study investigated whether 5-HT(2A) receptors were localized on dopamine cells within the A10 dopamine subnuclei of the rat, including the ventral tegmental area. We discovered that 5-HT(2A) receptor-like immunoreactivity colocalized with tyrosine hydroxylase, a marker for dopamine neurons, throughout the A10 dopamine cell population. Colocalization was most prominent in rostral and mid A10 regions, including the paranigral, parabrachial, and interfascicular subnuclei. Though more rare, non-dopaminergic neurons also expressed 5-HT(2A) receptor immunoreactivity in the ventral tegmental area. Additionally, although a dense population of 5-HT(2A) immunoreactive cells was observed in the rostral dorsal raphe nucleus, rarely were these cells immunoreactive for tyrosine hydroxylase. The linear raphe A10 dopamine subdivisions also displayed a low degree of 5-HT(2A) receptor and tyrosine hydroxylase colocalization. These findings provide an anatomical basis for the physiological modulation of dopamine neurons in the rostral ventral tegmental area either directly, by 5-HT(2A) receptors localized on dopamine cells, or indirectly, through a non-dopaminergic mechanism. Interestingly, 5-HT(2A) receptors were expressed on dopamine neurons in several A10 subnuclei that project to mesolimbic forebrain regions implicated in drug addiction, and recent evidence indicates that ventral tegmental area 5-HT(2A) receptor activation may modulate reward-related behavior in rodents. 5-HT(2A) receptors were also expressed on dopamine cells in A10 subnuclei that project to forebrain areas that have been implicated in schizophrenia, and atypical antipsychotic drugs have high affinities for 5-HT(2A) receptors. Thus, findings in this study could have important implications for understanding 5-HT and dopamine circuitry dysfunction in schizophrenia.

Evidence for the preferential involvement of 5-HT2A serotonin receptors in stress- and drug-induced dopamine release in the rat medial prefrontal cortex.

The mechanism(s) by which serotonin modulates dopamine release in the medial prefrontal cortex is not known, although studies suggest an involvement of 5-HT2 family receptors. We employed in vivo microdialysis and putatively selective 5-HT2A antagonists (M100907, MDL 11,939, SR46349B) to determine if 5-HT2A receptors are responsible for both drug- and stress-induced DA release in the medial prefrontal cortex. MDL 11,939 and SR46349B receptor-binding studies indicated, for the first time, that only MDL 11,939 had greater selectivity for the 5-HT2A vs the 5-HT2C receptor subtypes similar to M100907, and that both showed low or no affinity for non-5-HT2 receptors. Reverse dialysis with 5-HT2A antagonists had little or no effect on basal dopamine efflux. However, intracortical administration of MDL 11,939 or M100907 attenuated dopamine release induced by systemic administration of the 5-HT2 agonist DOI. Dual-probe microdialysis demonstrated that systemic DOI also increased glutamate concentrations in the ventral tegmental area (VTA). This was blocked by intracortical M100907. Cortical perfusion with M100907, or the atypical antipsychotic drug risperidone, but not the 5-HT2B/C ligand SB 206553, also decreased dopamine release induced physiologically by stress. These results indicate that stimulation of cortical 5-HT2A receptors increases the release of dopamine from the mesocortical system. They suggest that this effect may be mediated by increases in glutamate release from corticotegmental projections to the VTA. Additionally, they indicate that cortical 5-HT2A receptors modulate evoked dopamine release, such as that observed physiologically following mild stress. These findings may have implications for the pharmacological treatment of disorders resulting from or exacerbated by stress.

Effects of catecholamine uptake blockers in the caudate-putamen and subregions of the medial prefrontal cortex of the rat

Altered dopamine regulation in the medial prefrontal cortex has been linked to drug abuse and disorders such as schizophrenia. Heterogeneous expression of the dopamine transporter, as well as the ability of the norepinephrine transporter to clear dopamine in the prefrontal cortex, delineates two potential sites for the regulation of synaptic dopamine within the cortex. The present study used in vivo microdialysis to compare the effects of local infusions of dopamine and norepinephrine uptake blockers in the caudate putamen and two subregions of the prefrontal cortex, the anterior cingulate and prelimbic/infralimbic cortices. Results revealed that all dopamine uptake blockers produced greater increases in dopamine efflux in the caudate-putamen relative to the prefrontal cortex. In addition, amphetamine administration increased dopamine efflux to a greater degree in the prelimbic, relative to the anterior cingulate, cortex. In contrast, the increase in dopamine efflux was similar in both subregions in the presence of nomifensine and desmethylimipramine. Infusions of the selective dopamine uptake blocker GBR 12909 failed to alter dopamine efflux in any prefrontocortical subregion. These data indicate a more prominent role for the dopamine transporter in the clearance of extracellular dopamine in the caudate-putamen relative to the prefrontal cortex and an important role for NET in the clearance of dopamine in both the prelimbic and anterior cingulate subregions of the rat medial prefrontal cortex.

M100,907, a selective 5-HT2A antagonist, attenuates dopamine release in the rat medial prefrontal cortex

Previous research has suggested that serotonin 5-HT(2A) receptors modulate the functioning of the mesocortical dopamine (DA) pathway. However, the specific role of 5-HT(2A) receptors localized within the medial prefrontal cortex (mPFC) is not known. The present study employed in vivo microdialysis to examine the role of this receptor in the modulation of basal and K(+)-stimulated (Ca(2+)-dependent) DA release. The selective 5-HT(2A) antagonist M100,907 was infused directly into the mPFC of conscious rats. This resulted in a concentration-dependent blockade of K(+)-stimulated DA release. Intracortical application of M100,907 also blocked increases in DA release produced by the systemic administration of the 5-HT(2A/2C) agonist, 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI). These findings demonstrate that local 5-HT(2A) antagonism has an inhibitory effect on stimulated, Ca(2+)-dependent DA release. They suggest that cortical 5-HT(2A) receptors potentiate the phasic release of mesocortical DA.

A neurochemical heterogeneity of the rat striatum as measured by in vivo electrochemistry and microdialysis

The neurochemical heterogeneity of the rat striatum was assessed in vivo by measuring subregional changes in extracellular dopamine and DOPAC by in vivo electrochemistry and microdialysis in response to amphetamine and the D2 antagonist, (-)-sulpiride. Both in vivo electrochemical and microdialysis experiments indicated a significant rostrocaudal gradient in dopamine release following amphetamine. The increase in dopamine release was highest in the rostral areas (over 800% of baseline values) and lowest in the most caudal subregion (425% of baseline). No lateromedial differences in dopamine release were observed. DOPAC levels decreased in dialysates but were similar for all 6 subregions examined. In contrast, D2 blockade with (-)-sulpiride revealed a lateromedial gradient in the increases seen for dopamine and DOPAC such that greater increases were observed in the lateral subregions. (-)-Sulpiride did not produce any differential effects along the rostrocaudal axis. The regional gradients detected in extracellular fluid changes of dopamine and DOPAC indicate that dopamine release is locally regulated by an interaction between the density of dopaminergic innervation to a particular subregion and the D2 receptor density.

RET Is Dispensable for Maintenance of Midbrain Dopaminergic Neurons in Adult Mice

Glial cell-line derived neurotrophic factor (GDNF)-mediated RET tyrosine kinase signaling is implicated in the survival of several PNS and CNS neuronal populations that are important in the pathogenesis of several disorders including Parkinsons disease and drug addiction. However, it has been difficult to study these processes and the physiological importance of this pathway in adult mice because of the neonatal lethality of Gdnf and Ret null mice. We report successful creation of RET conditional reporter mice to investigate postnatal physiologic roles of RET and monitor the fate of RET-expressing cell types. To delete RET specifically in dopaminergic neurons and determine the physiologic requirement of RET in the maintenance of substantia nigra compacta (SNC) and ventral tegmental area (VTA), we bred the RET conditional mice with mice that specifically express Cre from the dopamine transporter (Dat) locus. A detailed morphometric and biochemical analysis including dopaminergic neuron number and size in SNC and VTA, and fiber density in the striatum and nucleus accumbens, and dopamine levels indicate that RET is not required for providing global trophic support to midbrain dopaminergic neurons in adult mice. Furthermore, RET deficiency in these neurons does not cause major sensorimotor abnormalities. Hence our results support the idea that RET signaling is not critical for the normal physiology of the SNC and VTA in adult mice.

Effects of cathinone and amphetamine on the neurochemistry of dopamine in vivo

The effects of (-)cathinone, the primary psychoactive alkaloid of the Khat plant, were compared to those of (+)amphetamine in the anterior caudate-putamen and the nucleus accumbens. In vivo microdialysis was used to measure extracellular levels of dopamine and metabolites in both regions of the brain simultaneously, after intraperitoneal administration of 0.8, 1.6 or 3.2 mg/kg of either drug (doses expressed as the salts). Both drugs increased levels of dopamine but decreased levels of metabolites in a dose-dependent manner. However, the relative magnitude of these effects depended upon the specific drug, the dose and area of the brain examined. At the largest dose used, amphetamine had a relatively greater effect than cathinone on dopamine in both caudate and accumbens. However, among smaller doses, this difference was only observed in the nucleus accumbens after administration of 1.6 mg/kg. The results also demonstrated a differential regional effect of both drugs at 3.2 mg/kg, in that both had a greater effect on dopamine in the caudate, as opposed to the accumbens. These findings demonstrate a functional heterogeneity of the striatum of the rat, that may be relevant to the understanding of both normal brain function and the neural responses to psychoactive drugs.

LRRK2-mediated neurodegeneration and dysfunction of dopaminergic neurons in a Caenorhabditis elegans model of Parkinson's disease.

Mutations in LRRK2 are thus far the most frequent known cause of autosomal dominant and idiopathic Parkinsons disease (PD) with prevalent mutations being found within the GTPase (R1441C/G) and kinase (G2019S) domains. Previous in vitro studies have revealed that R1441C and G2019S mutations are associated with increased kinase activity. To better understand LRRK2-linked PD pathogenesis in vivo, we have generated transgenic C. elegans overexpressing human LRRK2 wild type, R1441C and G2019S in dopaminergic (DA) neurons. Overexpression of these LRRK2 proteins causes age-dependent DA neurodegeneration, behavioral deficits, and locomotor dysfunction that are accompanied by a reduction of dopamine levels in vivo. In comparison, R1441C and G2019S mutants cause more severe phenotypes than the wild type protein. Interestingly, treatment with exogenous dopamine rescues the LRRK2-induced behavioral and locomotor phenotypes. In contrast, expression of the GTP binding defective mutant, K1347A, or knockout of the C. elegans LRRK2 homolog, LRK-1, prevents the LRRK2-induced neurodegeneration and behavioral abnormalities. Hence, our transgenic LRRK2 C. elegans models recapitulate key features of PD including progressive neurodegeneration, impairment of dopamine-dependent behavior and locomotor function, and reduction in dopamine levels. Furthermore, our findings provide strong support for the critical role of GTPase/kinase activity in LRRK2-linked pathologies. These invertebrate models will be useful for studying pathogenesis of PD and for development of potential therapeutics for the disease.

Local infusion of the serotonin antagonists ritanserin or ICS 205,930 increases in vivo dopamine release in the rat medial prefrontal cortex

Previous research has indicated that atypical antipsychotic drugs like clozapine preferentially increase dopamine (DA) release from the mesocortical, relative to the nigrostriatal, system. While these drugs generally have weak affinity for the D2 receptor subtype, they are potent antagonists of the 5‐hydroxytryptamine2 (5‐HT2) receptor. Research into neurotransmitter interactions indicates that 5‐HT modulates DA release, but the nature of this interaction may depend upon the specific 5‐HT receptor subtype and the neuronal location of that subtype. The present research tested the hypothesis that 5‐HT2 receptors localized near mesocortical DA nerve terminals regulate DA release. This was accomplished by infusing the specific 5‐HT2 antagonist ritanserin directly into the medial prefrontal cortex through reverse dialysis in vivo in the rat. Cortical extracellular fluid was then extracted by microdialysis and DA was subsequently assayed by HPLC with electrochemical detection. These results were compared to the systemic administration of ritanserin (1.0–5.0 mg/kg i.p.) and the local application of ICS 205,930, an antagonist at the 5‐HT3/4 receptor subtypes. Both 5‐HT antagonists increased cortical DA levels when infused locally at concentrations of 100 μM (12 nmoles/60 min), and these results were concentration‐dependent. Systemically administered ritanserin also dose‐dependently increased cortical DA efflux. These results indicate that atypical antipsychotic drugs may increase mesocortical DA release by antagonizing 5‐HT receptors located in the prefrontal cortex. Furthermore, 5‐HT may normally inhibit cortical DA release by actions at the 5‐HT2 receptor subtype.