Adenylyl cyclase is the prototypical second messenger generator. Nearly all of the eight cloned adenylyl cyclases are regulated by one or other arm of the phospholipase C pathway. Functional and ultrastructural investigations have shown that adenylyl cyclases are intimately associated with sites of calcium ion entry into the cell. Oscillations in cellular cyclic AMP levels are predicted to arise because of feedback inhibition of adenylyl cyclase by Ca2+. Such findings inextricably intertwine cellular signalling by cAMP and internal Ca2+ and extend the known regulatory modes available to cAMP.

Dopamine (DA)-containing projections to the cerebral cortex are considered to play an important role in cognitive processes. Using a recently developed monoclonal antiserum directed against DA and an antibody directed against tyrosine hydroxylase in combination with Golgi impregnation and electron microscopy, we have observed that DA and tyrosine hydroxylase afferents establish symmetric membrane specializations with the soma, dendritic shafts, and spines of identified pyramidal cells in the prefrontal, cingulate, and motor cortex of primates. The axospinous contacts invariably formed part of a synaptic complex in which the dendritic spine of a pyramidal neuron was the target of both a DApositive symmetric and an unlabeled asymmetric bouton. This arrangement allows direct DA modulation of the overall excitability of cortical projection neurons by altering local spine responses to excitatory inputs. Dopamine (DA) has been implicated in a wide range of cognitive and affective behaviors and its importance for diseases affecting thought processes, such as schizophrenia, has been repeatedly stressed (1). Although the highest brain concentrations of DA are found in the caudate nucleus, this neurotransmitter is also present in the cerebral cortex, particularly in areas like the prefrontal cortex (2, 3) that are involved in emotional and cognitive processing. In rodents (4) and nonhuman primates (5), experimental depletion of DA has, in fact, been shown to result in cognitive deficits.A more complete understanding of DA's role in cortical function will require detailed knowledge about the cortical targets of DA afferents. The DA innervation of the primate prefrontal cortex has so far been studied only at the light microscopic level (6, 7). These studies reveal rich plexuses of DA-containing fibers in specific layers of the prefrontal cortex in monkeys (6, 7) and humans (8). However, definitive information on the postsynaptic structures innervated by these fibers is lacking. Accordingly, the goal of the present study was to provide ultrastructural data on the nature, distribution, and postsynaptic targets of dopaminergic boutons in the primate neocortex. We have used a recently available monoclonal antiserum directed against DA developed in one of our laboratories (9) as well as an antibody to its rate-limiting enzyme, tyrosine hydroxylase (TH) (10), and combined these methods with Golgi impregnation and electron microscopy (EM) to visualize DA synapses in the primate cortex. MATERIALS AND METHODSFive adult male rhesus monkey (Macaca mulatta) were deeply anesthetized and transcardially perfused using the fixative of Van Eden et al. (11) for DA and that of Somogyi and Takagi (12) for TH. After perfusion, blocks of tissue were taken from the dorsal bank of the principal sulcus (Walker's area 46), the anterior cingulate gyrus (Brodmann's area 24), and the primary motor cortex (Brodmann's area 4) (see Fig. 1) and were then sectioned perpendicular to the pial surface on a vibratome or cryostat. The sections were stai...

Posttraumatic stress disorder (PTSD) is characterized by a hypermnesia of the trauma and by a memory impairment that decreases the ability to restrict fear to the appropriate context. Infusion of glucocorticoids in the hippocampus after fear conditioning induces PTSD-like memory impairments and an altered pattern of neural activation in the hippocampal-amygdalar circuit. Mice become unable to identify the context as the correct predictor of the threat and show fear responses to a discrete cue not predicting the threat in normal conditions. These data demonstrate PTSD-like memory impairments in rodents and identify a potential pathophysiological mechanism of this condition.

Extinction of classical fear conditioning is thought to involve activity-dependent potentiation of synaptic transmission in the medial prefrontal cortex (mPFC), resulting in the inhibition of amygdala-dependent fear responses. While many studies have addressed the mechanisms underlying extinction learning, it is unclear what determines whether extinction memory is consolidated or whether spontaneous recovery of the fear response occurs. Here we show, using a combined electrophysiological and immunocytochemical approach, that spontaneous recovery of conditioned fear in mice is associated with a prolonged expression of long-term depression of synaptic transmission in the mPFC and the failure of induction of the immediate-early genesc-Fos and zif268 in the mPFC and the basolateral nucleus of the amygdala. This suggests that coordinated activity-dependent changes in gene expression in the mPFC and the amygdala may underlie the formation of long-term fear extinction memory.

Whereas the neuronal substrates underlying the acquisition of auditory fear conditioning have been widely studied, the substrates and mechanisms mediating the acquisition of fear extinction remain largely elusive. Previous reports indicate that consolidation of fear extinction depends on the mitogen-activated protein kinase/extracellular-signal regulated kinase (MAPK/ERK) signalling pathway and on protein synthesis in the medial prefrontal cortex (mPFC). Based on experiments using the fear-potentiated startle paradigm suggesting a role for neuronal plasticity in the basolateral amygdala (BLA) during fear extinction, we directly addressed whether MAPK/ERK signalling in the basolateral amygdala is necessary for the acquisition of fear extinction using conditioned freezing as a read-out. First, we investigated the regional and temporal pattern of MAPK/ERK activation in the BLA following extinction learning in C57Bl/6J mice. Our results indicate that acquisition of extinction is associated with an increase of phosphorylated MAPK/ERK in the BLA. Moreover, we found that inhibition of the MAPK/ERK signalling pathway by intrabasolateral amygdala infusion of the MEK inhibitor, U0126, completely blocks acquisition of extinction. Thus, our results indicate that the MAPK/ERK signalling pathway is required for extinction of auditory fear conditioning in the BLA, and support a role for neuronal plasticity in the BLA during the acquisition of fear extinction.

Ca2ϩ -sensitive adenylyl cyclases provide a key point for integration of signaling by [Ca 2ϩ ] i 1 and cAMP (1). Their likely contribution to cellular regulation is underscored by the fact that whether they are expressed heterologously or endogenously, these cyclases are regulated by physiological transitions in [Ca 2ϩ ] i (2-9). Somewhat unexpectedly, the Ca 2ϩ -sensitive adenylyl cyclases, whether they are expressed endogenously or heterologously, show a preference for regulation by Ca 2ϩ entering the cell over Ca 2ϩ released from intracellular stores (7, 10). Even more strikingly, Ca 2ϩ entry promoted by ionophore is unable to regulate transfected Ca 2ϩ -stimulable adenylyl cyclases (7). Consequently, we had proposed that Ca 2ϩ -stimulable adenylyl cyclases and capacitative Ca 2ϩ entry channels (I CRAC s) 2 were functionally colocalized (7). However, it is always conceivable that when they are transfected, adenylyl cyclases are expressed in discrete cellular domains, which reflects the response of the cell to overexpression of signaling molecules. It is therefore of considerable interest to determine whether similar colocalization is encountered with endogenously expressed adenylyl cyclase in continuous cell lines, which are more appropriate models of a normal signaling repertoire. Previous studies have established that the endogenous Ca 2ϩ -inhibitable adenylyl cyclase, which is the predominant form in C6 -2B glioma cells (11), is also regulated by the entry of Ca 2ϩ rather than release from intracellular stores, which was triggered by a variety of treatments (10). However, it is not known whether such endogenous adenylyl cyclases show a similar absolute dependence on capacitative versus any other form of entry. Such a requirement would predict a close association between entry sites and the cyclases. The potential intimacy of an endogenous Ca 2ϩ -inhibitable adenylyl cyclase and Ca 2ϩ entry channels is explored in the present study by assessing (i) the sensitivity of the cyclase to various types of [Ca 2ϩ ] i rise, (ii) whether this action can be differentially modulated by fast versus slow chelators of Ca 2ϩ , (iii) the role of the cytoskeleton, and (iv) whether the simple activity of the I CRAC , independent of the ion being transported, can modulate the enzyme. Cell Culture-C6 -2B rat glioma cells were maintained in 13 ml of F-10 medium (Life Technologies, Inc.) with 10% (v/v) bovine calf serum (Gemini) in 75-cm 2 flasks at 37°C in a humidified atmosphere of 95% air and 5% CO 2 . Cells were plated at approximately 70% confluency in 24-well plates for cAMP accumulation experiments. EXPERIMENTAL PROCEDURES MaterialsMeasurement of cAMP Accumulation-cAMP accumulation in intact cells was measured according to the method of Evans et al. (12) as described previously (7) with some modifications. C6 -2B cells on 24-well plates were incubated in F-10 medium (60 min at 37°C) with [2-3 H] adenine (1.5 Ci/well) to label the ATP pool. The cells were then washed once and incubated with a nominally Ca 2ϩ -free Kre...

Fear conditioning is a popular model for investigating physiological and cellular mechanisms of memory formation. In this paradigm, a footshock is either systematically associated to a tone (paired conditioning) or is pseudorandomly distributed (unpaired conditioning). In the former procedure, the tone/shock association is acquired, whereas in the latter procedure, the context/shock association will prevail. Animals with chronically implanted recording electrodes show enhanced amplitude of the extracellularly recorded field EPSP in CA1 pyramidal cells for up to 24 h after unpaired, but not paired, fear conditioning. This is paralleled by a differential activation of the ERK/CREB pathway in CA1, which is monophasic in paired conditioning (0-15 min post-conditioning), but biphasic (0-1 h and 9-12 h post-conditioning) in unpaired conditioning as revealed by immunocytochemistry and Western blotting. Intrahippocampal injection of the MEK inhibitor U0126 prior to each phase prevents the activation of both ERK1/2 and CREB after unpaired conditioning. Block of any activation phase leads to memory impairment. We finally reveal that the biphasic activation of ERK/CREB activity is independently regulated, yet both phases are critically required for the consolidation of long-term memories following unpaired fear conditioning. These data provide compelling evidence that CA1 serves different forms of memory by expressing differential cellular mechanisms that are dependent on the training regime.

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