The serotonin system is proposed to regulate physiology and behavior and to underlie mood disorders; nevertheless, the circuitry controlling serotonergic neurons remains uncharacterized. We therefore generated a comprehensive whole-brain atlas defining the monosynaptic inputs onto forebrain-projecting serotonergic neurons of dorsal versus median raphe based on a genetically restricted transsynaptic retrograde tracing strategy. We identified discrete inputs onto serotonergic neurons from forebrain and brainstem neurons, with specific inputs from hypothalamus, cortex, basal ganglia, and midbrain, displaying a greater than anticipated complexity and diversity in cell-type-specific connectivity. We identified and functionally confirmed monosynaptic glutamatergic inputs from prefrontal cortex and lateral habenula onto serotonergic neurons as well as a direct GABAergic input from striatal projection neurons. In summary, our findings emphasize the role of hyperdirect inputs to serotonergic neurons. Cell-type-specific classification of connectivity patterns will allow for further functional analysis of the diverse but specific inputs that control serotonergic neurons during behavior.

The influx of calcium into the postsynaptic neuron is likely to be an important event in memory formation. Among the mechanisms that nerve cells may use to alter the time course or size of a spike of intracellular calcium are cytosolic calcium binding or "buffering" proteins. To consider the role in memory formation of one of these proteins, calbindin D28K, which is abundant in many neurons, including the CAl pyramidal cells of the hippocampus, transgenic mice deficient in calbindin D28K have been created. These mice show selective impairments in spatial learning paradigms and fail to maintain long-term potentiation. These results suggest a role for calbindin D28K protein in temporally extending a neuronal calcium signal, allowing the activation of calciumdependent intracellular signaling pathways underlying memory function.

Caffeine has been imbibed since ancient times in tea and coffee, and more recently in colas. Caffeine owes its psychostimulant action to a blockade of adenosine A(2A) receptors, but little is known about its intracellular mechanism of action. Here we show that the stimulatory effect of caffeine on motor activity in mice was greatly reduced following genetic deletion of DARPP-32 (dopamine- and cyclic AMP-regulated phosphoprotein of relative molecular mass 32,000). Results virtually identical to those seen with caffeine were obtained with the selective A(2A) antagonist SCH 58261. The depressant effect of the A(2A) receptor agonist, CGS 21680, on motor activity was also greatly attenuated in DARPP-32 knockout mice. In support of a role for DARPP-32 in the action of caffeine, we found that, in striata of intact mice, caffeine increased the state of phosphorylation of DARPP-32 at Thr 75. Caffeine increased Thr 75 phosphorylation through inhibition of PP-2A-catalysed dephosphorylation, rather than through stimulation of cyclin-dependent kinase 5 (Cdk5)-catalysed phosphorylation, of this residue. Together, these studies demonstrate the involvement of DARPP-32 and its phosphorylation/dephosphorylation in the stimulant action of caffeine.

The two mitogen-activated protein kinases (MAPKs), extracellular signal-regulated protein kinase 1 and 2 (ERK1/2), are involved in the control of gene expression via phosphorylation and activation of the transcription factors cyclic AMP response element binding protein (CREB) and Elk-1. Here, we have examined the effect of haloperidol and clozapine, two antipsychotic drugs, and eticlopride, a selective dopamine D 2 receptor antagonist, on the state of phosphorylation of ERK1/2, CREB and Elk-1, in the mouse dorsal striatum. Administration of the typical anti-psychotic haloperidol stimulated the phosphorylation of ERK1/2, CREB and Elk-1. Virtually identical results were obtained using eticlopride. In contrast, the atypical anti-psychotic clozapine reduced ERK1/2, CREB and Elk-1 phosphorylation. This opposite regulation was specifically exerted by haloperidol and clozapine on ERK, CREB, and Elk-1 phosphorylation, as both anti-psychotic drugs increased the phosphorylation of the dopamine-and cyclic AMPregulated phosphoprotein of 32 kDa (DARPP-32) at the cyclic AMP-dependent protein kinase (PKA) site. The activation of CREB and Elk-1 induced by haloperidol appeared to be achieved via different signalling pathways, as inhibition of ERK1/2 activation abolished the stimulation of Elk-1 phosphorylation without affecting CREB phosphorylation. This study shows that haloperidol and clozapine induce distinct patterns of phosphorylation in the dorsal striatum. The results provide a novel biochemical paradigm elucidating the molecular mechanisms underlying the distinct therapeutic actions of typical and atypical anti-psychotic agents. Treatment with anti-psychotic (or neuroleptic) drugs currently represents the most common therapy for schizophrenia. One major limitation in the use of conventional antipsychotic drugs, such as haloperidol, is that their prolonged administration induces extra-pyramidal side-effects by modifying transmission in the basal ganglia, a group of brain structures involved in the control of voluntary movements. The atypical anti-psychotic, clozapine, has a superior therapeutic efficacy and does not produce extra-pyramidal sideeffects. The use of this drug, however, is complicated by the relatively high incidence of agranulocytosis, a condition that requires the immediate suspension of clozapine administration.A common feature of anti-psychotic drugs is their ability to act as dopamine D 2 receptor antagonists. Blockade of dopamine D 2 receptors profoundly affects gene expression in the striatum, the major receiving area of the basal ganglia.Administration of haloperidol produces a rapid increase in the expression of various genes including the immediate early gene c-fos (Dragunow et al. 1990;Miller 1990) Abbreviations used: CREB, cyclic AMP response element binding protein; DARPP-32, dopamine and cyclic AMP-regulated phosphoprotein of 32 kDa; ERK1/2, extracellular signal-regulated protein kinase 1 and 2; MAPKs, mitogen-activated protein kinases; MEK, MAPK/ERK kinase.