Morphometric characterization of synapses in the primate prefrontal cortex formed by afferents from the mediodorsal thalamic nucleus.

The main thalamic afferentation of the prefrontal cortex (PFC) originates in the mediodorsal nucleus (MD). Although it is suggested that this pathway is affected in schizophrenia, there is a lack of functional and structural data regarding its synaptic organization. The scope of this study was to characterize the ultrastructural features of thalamocortical synapses formed by afferents from the MD by applying anterograde tract tracing, immunohistochemical detection of parvalbumin (PV, a probable marker of thalamocortical endings), and quantitative electron microscopic techniques to the PFC of the macaque monkey. Our findings indicate that anterogradely-labeled and PV-immunoreactive boutons exhibit similar ultrastructural properties, characterized by their larger size, higher incidence of release sites and a higher occurrence of mitochondria when compared to non-labeled, excitatory-like endings in the middle layers of the PFC. Although most of the contacts were made on spines in both cases, PV-immunopositive axon terminals apparently targeted dendritic shafts at about twice the frequency found for anterogradely-labeled afferents from the MD (20.5% and 9.5%, respectively). This result suggests diversity among thalamocortical and/or PV-immunoreactive axon terminals of the PFC. In accordance with studies in other cortical areas, our findings suggest that corollary discharge through the mediodorsal thalamocortical projection is also adapted to synaptic transmission with high efficacy and probably exhibits marked short-term temporal dynamics in the PFC.

Differential anterior prefrontal activation during the recognition stage of a spatial working memory task.

Neuroimaging studies commonly show widespread activations in the prefrontal cortex during various forms of working memory and long-term memory tasks. However, the anterior prefrontal cortex (aPFC, Brodmann area 10) has been mainly associated with retrieval in episodic memory, and its role in working memory is less clear. We conducted an event-related functional magnetic resonance imaging study to examine brain activations in relation to recognition in a spatial delayed-recognition task. Similar to the results from previous findings, several frontal areas were strongly activated during the recognition phase of the task, including the aPFC, the lateral PFC and the anterior cingulate cortex. Although the aPFC was more active during the recognition phase, it was also active during the delay phase of the spatial working memory task. In addition, the aPFC showed greater activity in response to negative probes (non-targets) than to positive probes (targets). While our analyses focused on examining signal changes in the aPFC, other prefrontal regions showed similar effects and none of the areas were more active in response to the positive probes than to the negative probes. Our findings support the conclusion that the aPFC is involved in working memory and particularly in processes that distinguish target and non-target stimuli during recognition.

Division of labor among distinct subtypes of inhibitory neurons in a cortical microcircuit of working memory.

A conspicuous feature of cortical organization is the wide diversity of inhibitory interneurons; their differential computational functions remain unclear. Here we propose a local cortical circuit in which three major subtypes of interneurons play distinct roles. In a model designed for spatial working memory, stimulus tuning of persistent activity arises from the concerted action of widespread inhibition mediated by perisoma-targeting (parvalbumin-containing) interneurons and localized disinhibition of pyramidal cells via interneuron-targeting (calretinin-containing) interneurons. Moreover, resistance against distracting stimuli (a fundamental property of working memory) is dynamically controlled by dendrite-targeting (calbindin-containing) interneurons. The experimental observation of inverted tuning curves of monkey prefrontal neurons recorded during working memory supports a key model prediction. This work suggests a framework for understanding the division of labor and cooperation among different inhibitory cell types in a recurrent cortical circuit.

Sustained mnemonic response in the human middle frontal gyrus during on-line storage of spatial memoranda.

The mapping of cognitive functions to neural systems is a central goal of cognitive neuroscience. On the basis of homology with lesion and physiological studies in nonhuman primates, Brodmann's area (BA) 46/9 in the middle frontal gyrus (MFG) has been proposed as the cortical focus for both the storage as well as processing components of working memory in the human brain, but the evidence on the segregation of these components and their exact areal localization has been inconsistent. In order to study this issue and increase the temporal resolution of functional mapping, we disambiguated the storage component of working memory from sensory and motor responses by employing functional magnetic resonance imaging (fMRI) in spatial delayed-response (DR) tasks with long delay intervals and different conditions of demand. We here show that BA 46 can support a sustained mnemonic response for as long as 24 sec in a high-demand task and the signal change in this area exceeded that in the other prefrontal areas examined. Our findings support a conservation of functional architecture between human and nonhuman primate in showing that the MFG is prominently engaged in the storage of spatial information.

Distribution of protein phosphatases-1 alpha and -1 gamma 1 and the D(1) dopamine receptor in primate prefrontal cortex: Evidence for discrete populations of spines.

The function of G protein-coupled receptors depends on the availability of the appropriate signal transduction proteins in close proximity to the receptor. We have examined and quantified in primate prefrontal cortex the subcellular distribution of two isoforms of protein phosphatase-1 (PP1), PP1 alpha and PP1 gamma 1, which are components of the signal transduction pathway accessed by the D(1) dopamine receptor. Both PP1 alpha- and PP1 gamma 1-labeled puncta are seen in cortex, basal ganglia, hippocampus, and thalamus. Viewed with the electron microscope, both PP1 isoforms are selectively localized to dendritic spines and are found in different percentages of spines; PP1 alpha is present in roughly 70% and PP1 gamma 1 in roughly 40% of dendritic spines. Our analysis indicates that three populations of spines are defined by the distribution of these PP1 isoforms: those that contain both PP1 alpha and PP1 gamma 1, those that contain only PP1 alpha and those that contain neither. The D(1) receptor is present in a subset of the population that contains both PP1 alpha and PP1 gamma 1. The nonhomogeneous distribution of signal transduction proteins in the spines and dendrites of cortical pyramidal cells may help to explain differences in the actions of receptors that nominally use the same signal-transduction pathway.

Primate rhinal cortex participates in both visual recognition and working memory tasks: functional mapping with 2-DG.

The rhinal cortex in the medial temporal lobe has been implicated in object recognition memory tasks and indeed is considered to be the critical node in a visual memory network. Previous studies using the 2-deoxyglucose method have shown that thalamic and hippocampal structures thought to be involved in visual recognition memory are also engaged by spatial and object working memory tasks in the nonhuman primate. Networks engaged in memory processing can be recognized by analysis of patterns of activation accompanying performance of specifically designed tasks. In the present study, we compared metabolic activation of the entorhinal and perirhinal cortex during the performance of three working memory tasks [delayed response (DR), delayed alternation (DA), and delayed object alternation (DOA)] to that induced by a standard recognition memory task [delayed match-to-sample (DMS)] and a sensorimotor control task in rhesus monkeys. A region-of-interest analysis revealed elevated local cerebral glucose utilization in the perirhinal cortex in animals performing the DA, DOA, and DMS tasks, and animals performing the DMS task were distinct in showing a strong focus of activation in the lateral perirhinal cortex. No significant differences were evident between groups performing memory and control tasks in the entorhinal cortex. These findings suggest that the perirhinal cortex may play a much broader role in memory processing than has been previously thought, encompassing explicit working memory as well as recognition memory.

Muscarinic m1 and m2 receptor proteins in local circuit and projection neurons of the primate striatum: anatomical evidence for cholinergic modulation of glutamatergic prefronto-striatal pathways.

The cellular and subcellular localization of muscarinic receptor proteins m1 and m2 was examined in the neostriatum of macaque monkeys by using light and electron microscopic immunocytochemical techniques. Double-labeling immunocytochemistry revealed m1 receptors in calbindin-D28k--positive medium spiny projection neurons. Muscarinic m1 labeling was dramatically more intense in the striatal matrix compartment in juvenile monkeys but more intense in striosomes in the adult caudate, suggesting that m1 expression undergoes a developmental age-dependent change. Ultrastructurally, m1 receptors were predominantly localized in asymmetric synapse-forming spines, indicating that these spines receive extrastriatal excitatory afferents. The association of m1-positive spines with lesion-induced degenerating prefronto-striatal axon terminals demonstrated that these afferents originate in part from the prefrontal cortex. The synaptic localization of m1 in these spines indicates a role of m1 in the modulation of excitatory neurotransmission. To a lesser extent, m1 was present in symmetric synapses, where it may also modulate inhibitory neurotransmission originating from local striatal neurons or the substantia nigra. Conversely, m2/choline acetyltransferase (ChAT) double labeling revealed that m2-positive neurons corresponded to large aspiny cholinergic interneurons and ultrastructurally, that the majority of m2 labeled axons formed symmetric synapses. The remarkable segregation of the m1 and m2 receptor proteins to projection and local circuit neurons suggests a functional segregation of m1 and m2 mediated cholinergic actions in the striatum: m1 receptors modulate extrinsic glutamatergic and monoaminergic afferents and intrinsic GABAergic afferents onto projection neurons, whereas m2 receptors regulate acetylcholine release from axons of cholinergic interneurons.

Prefrontal microcircuits: membrane properties and excitatory input of local, medium, and wide arbor interneurons.

To elucidate cortical mechanisms involved in higher cortical functions such as working memory, we have examined feedforward excitation transmitted by identified pyramidal cells to interneurons with predominantly horizontal axonal arbors, using dual somatic recordings in prefrontal cortical slices. Interneurons with local (narrow) axonal arbors, especially chandelier interneurons, exhibited extremely narrow action potentials and high evoked firing rates, whereas neurons identified with wide arbor axons generated wider spikes and lower evoked firing rates with considerable spike adaptation, resembling that of pyramidal cells. Full reconstruction of differentially labeled neuronal pairs revealed that local arbor cells generally received a single but functionally reliable putative synaptic input from the identified pyramidal neuron member of the pair. In contrast, more synapses (two to five) were necessary to depolarize medium and wide arbor neurons reliably. The number of putative synapses and the amplitude of the postsynaptic response were remarkably highly correlated within each class of local, medium, and wide arbor interneurons (r = 0.88, 0.95, and 0.99, respectively). Similarly strong correlations within these subgroups were also present between the number of putative synapses and variance in the EPSP amplitudes, supporting the validity of our morphological analysis. We conclude that interneurons varying in the span of their axonal arbors and hence in the potential regulation of different numbers of cortical modules differ also in their excitatory synaptic input and physiological properties. These findings provide insight into the circuit basis of lateral inhibition and functional interactions within and between cortical columns in the cerebral cortex.

Coding specificity in cortical microcircuits: a multiple-electrode analysis of primate prefrontal cortex.

Neurons with directional specificities are active in the prefrontal cortex (PFC) during tasks that require spatial working memory. Although the coordination of neuronal activity in PFC is thought to be maintained by a network of recurrent connections, direct physiological evidence regarding such networks is sparse. To gain insight into the functional organization of the working memory system in vivo, we recorded simultaneously from multiple neurons spaced 0.2-1 mm apart in monkeys performing an oculomotor delayed response task. We used cross-correlation analysis and characterized the effective connectivity between neurons in relation to their spatial and temporal response properties. The majority of narrow (<5 msec) cross-correlation peaks indicated common input and were most often observed between pairs of neurons within 0.3 mm of each other. Neurons recorded at these distances represented the full range of spatial locations, suggesting that the entire visual hemifield is represented in modules of corresponding dimensions. Nearby neurons could be activated in any epoch of the behavioral task (stimulus presentation, delay, response). The incidence and strength of cross-correlation, however, was highest among cells sharing similar spatial tuning and similar temporal profiles of activation across task epochs. The dependence of correlated discharge on the functional properties of neurons was observed both when we analyzed firing from the task period as well as from baseline fixation. Our results suggest that the coding specificity of individual neurons extends to the local circuits of which they are part.

Dopamine D2 and D3 receptors are linked to the actin cytoskeleton via interaction with filamin A.

We have used a yeast two-hybrid approach to uncover protein interactions involving the D2-like subfamily of dopamine receptors. Using the third intracellular loop of the D2S and D3 dopamine receptors as bait to screen a human brain cDNA library, we identified filamin A (FLN-A) as a protein that interacts with both the D2 and D3 subtypes. The interaction with FLN-A was specific for the D2 and D3 receptors and was independently confirmed in pull-down and coimmunoprecipitation experiments. Deletion mapping localized the dopamine receptor-FLN-A interaction to the N-terminal segment of the D2 and D3 dopamine receptors and to repeat 19 of FLN-A. In cultures of dissociated rat striatum, FLN-A and D2 receptors colocalized throughout neuronal somata and processes as well as in astrocytes. Expression of D2 dopamine receptors in FLN-A-deficient M2 melanoma cells resulted in predominant intracellular localization of the D2 receptors, whereas in FLN-A-reconstituted cells, the D2 receptor was predominantly localized at the plasma membrane. These results suggest that FLN-A may be required for proper cell surface expression of the D2 dopamine receptors. Association of D2 and D3 dopamine receptors with FLN-A provides a mechanism whereby specific dopamine receptor subtypes may be functionally linked to downstream signaling components via the actin cytoskeleton.

The sensory nature of mnemonic representation in the primate prefrontal cortex.

A long-standing issue concerning the function of the primate dorsolateral prefrontal cortex is whether the activity of prefrontal neurons reflects the perceived sensory attributes of a remembered stimulus, or the decision to execute a motor response. To distinguish between these possibilities, we recorded neuronal activity from monkeys trained to make a saccade toward the brighter of two memoranda, under conditions of varied luminance. Our results indicated that during the delay period when sensory information was no longer available, neuronal discharge was modulated by the luminance of the stimulus appearing in the receptive field, and was directly correlated with psychophysical performance in the task. The findings suggest that although prefrontal cortex codes for a diversity of representations, including the decision for an impending response, a population of neurons maintains the dimensional attributes of remembered stimuli throughout the delay period, which allows for flexibility in the outcome of a mnemonic process.

Antipsychotic treatment induces alterations in dendrite- and spine-associated proteins in dopamine-rich areas of the primate cerebral cortex.

BACKGROUND: Mounting evidence indicates that long-term treatment with antipsychotic medications can alter the morphology and connectivity of cellular processes in the cerebral cortex. The cytoskeleton plays an essential role in the maintenance of cellular morphology and is subject to regulation by intracellular pathways associated with neurotransmitter receptors targeted by antipsychotic drugs. METHODS: We have examined whether chronic treatment with the antipsychotic drug haloperidol interferes with phosphorylation state and tissue levels of a major dendritic cytoskeleton-stabilizing agent, microtubule-associated protein 2 (MAP2), as well as levels of the dendritic spine-associated protein spinophilin and the synaptic vesicle-associated protein synaptophysin in various regions of the cerebral cortex of rhesus monkeys. RESULTS: Among the cortical areas examined, the prefrontal, orbital, cingulate, motor, and entorhinal cortices displayed significant decreases in levels of spinophilin, and with the exception of the motor cortex, each of these regions also exhibited increases in the phosphorylation of MAP2. No changes were observed in either spinophilin levels or MAP2 phosphorylation in the primary visual cortex. Also, no statistically significant changes were found in tissue levels of MAP2 or synaptophysin in any of the cortical regions examined. CONCLUSIONS: Our findings demonstrate that long-term haloperidol exposure alters neuronal cytoskeleton- and spine-associated proteins, particularly in dopamine-rich regions of the primate cerebral cortex, many of which have been implicated in the psychopathology of schizophrenia. The ability of haloperidol to regulate cytoskeletal proteins should be considered in evaluating the mechanisms of both its palliative actions and its side effects.

An astroglia-linked dopamine D2-receptor action in prefrontal cortex.

Typical neuroleptic drugs elicit their antipsychotic effects mainly by acting as antagonists at dopamine D2 receptors. Much of this activity is thought to occur in the cerebral cortex, where D2 receptors are found largely in inhibitory GABAergic neurons. Here we confirm this localization at the electron microscopic level, but additionally show that a subset of cortical interneurons with low or undetectable expression of D2 receptor isoforms are surrounded by astrocytic processes that strongly express D2 receptors. Ligand binding of isolated astrocyte preparations indicate that cortical astroglia account for approximately one-third of the total D2 receptor binding sites in the cortex, a proportion that we found conserved among rodent, monkey, and human tissues. Further, we show that the D2 receptor-specific agonist, quinpirole, can induce Ca(2+) elevation in isolated cortical astrocytes in a pharmacologically reversible manner, thus implicating this receptor in the signaling mechanisms by which astrocytes communicate with each other as well as with neurons. The discovery of D2 receptors in astrocytes with a selective anatomical relationship to interneurons represents a neuron/glia substrate for cortical dopamine action in the adult cerebral cortex and a previously unrecognized site of action for antipsychotic drugs with affinities at the D2 receptor.

Presynaptic regulation of recurrent excitation by D1 receptors in prefrontal circuits.

The prefrontal cortex plays a fundamental role in the working memory functions of the cerebral cortex and is also the site of dysfunction in several neurological and psychiatric disorders, including schizophrenia. Prefrontal neurons are distinguished by their capacity for sustained activity during the time a stimulus is held in memory, and this mnemonic response is considered a substrate for a variety of cognitive functions. The neuronal basis for sustained activity in prefrontal neurons is unknown but is thought to involve recurrent excitation among pyramidal neurons. Recent studies in awake behaving monkeys have demonstrated that the persistent activity in prefrontal neurons is modulated by dopamine. To examine the mechanisms by which dopamine might modulate transmission in local excitatory circuits, we have performed dual whole-cell recordings in connected pyramidal cell pairs with and without dopamine application. We find that dopamine reduces the efficacy of unitary excitatory neurotransmission in layer V pyramidal cells by decreasing its reliability. These effects, which are reproduced by a selective D1 agonist and blocked by a D1 antagonist, are independent of voltage changes and are not attenuated by blockade of sodium and potassium channels in the postsynaptic neurons. We conclude that attenuation of local horizontal excitatory synaptic transmission in layer V pyramidal neurons by dopamine is through D1 actions at a presynaptic site.

Verbal processing deficits in schizophrenia.

The authors reported that a subgroup of schizophrenic patients performed well on a tone serial position task but was impaired on an auditory word serial position task (Wexler, Stevens, Bowers, Cerniak, & Goldman-Rakic, 1998). This study assessed 30 schizophrenic and 32 controls (matched for comparable tone discrimination) on 4 versions of the verbal serial position tasks and 2 tone serial position tasks. Patients performed poorly on all verbal tasks but performed comparably to controls when tones served as stimuli. Proactive interference and visual presentation further compounded the verbal deficits. Deficits persisted with pronounceable nonword stimuli. These findings provide evidence of specific deficits in language-related processing, although the authors could not rule out the possibility that the differential effects that were observed between the tone and word tasks, and particularly among the verbal tasks, may result from differing discriminating power of the different tests.

Synaptic mechanisms and network dynamics underlying spatial working memory in a cortical network model.

Single-neuron recordings from behaving primates have established a link between working memory processes and information-specific neuronal persistent activity in the prefrontal cortex. Using a network model endowed with a columnar architecture and based on the physiological properties of cortical neurons and synapses, we have examined the synaptic mechanisms of selective persistent activity underlying spatial working memory in the prefrontal cortex. Our model reproduces the phenomenology of the oculomotor delayed-response experiment of Funahashi et al. (S. Funahashi, C.J. Bruce and P.S. Goldman-Rakic, Mnemonic coding of visual space in the monkey's dorsolateral prefrontal cortex. J Neurophysiol 61:331-349, 1989). To observe stable spontaneous and persistent activity, we find that recurrent synaptic excitation should be primarily mediated by NMDA receptors, and that overall recurrent synaptic interactions should be dominated by inhibition. Isodirectional tuning of adjacent pyramidal cells and interneurons can be accounted for by a structured pyramid-to-interneuron connectivity. Robust memory storage against random drift of the tuned persistent activity and against distractors (intervening stimuli during the delay period) may be enhanced by neuromodulation of recurrent synapses. Experimentally testable predictions concerning the neural basis of working memory are discussed.

Serotonin induces EPSCs preferentially in layer V pyramidal neurons of the frontal cortex in the rat.

The effect of serotonin (5-HT) on the release of glutamate was examined in pyramidal cells in layers II-VI of the frontal cortex. The intracellular recording electrode contained 1% biocytin so the neurons could later be visualized with an avidin-biotin peroxidase method. Pyramidal cells in layer V of the frontal cortex showed the greatest 5-HT-induced increase in both the frequency and amplitude of 'spontaneous' (non-electrically evoked) excitatory post-synaptic currents (EPSCs). A small proportion of neurons in layer II/III showed an increase in EPSC frequency, whereas cells in layer VI showed no significant change in either EPSC frequency or amplitude. The physiological response to 5-HT mirrors the high density of 5-HT(2A) receptors in layer V, as well as the pattern of thalamic projections in frontal cortex. The specific induction of EPSCs in layer V neurons suggests that 5-HT preferentially modulates the output neurons of the frontal cortex.

Prefrontal activation evoked by infrequent target and novel stimuli in a visual target detection task: an event-related functional magnetic resonance imaging study.

An event-related functional magnetic resonance imaging study of prefrontal cortex was conducted during which subjects performed a visual "oddball" target detection task. Exemplars of three stimulus categories were presented at a rate of one per 1.5 sec for 10 runs, each consisting of 132 trials. Standards were color squares of varying sizes that were presented on approximately 92% of trials. Targets were color circles of varying sizes presented irregularly on approximately 4% of trials. Novels were pictures of everyday objects that were also presented irregularly on approximately 4% of trials. Ten subjects participated in two separate sessions in which they were required to count mentally or to push a button whenever a target appeared. Targets evoked activation within prefrontal cortex, primarily within the middle frontal gyri (MFG). This MFG activation did not differ as a function of the required response. Novels did not evoke significant activity within this region despite evidence from a separate behavioral and event-related potential study demonstrating their strong influence on processing. In additional imaging sessions with two subjects, the rules were reversed to require a button press whenever an object, but not a circle, appeared. These former novels now evoked activation in the MFG, but the former target circles did not. These experiments indicate that MFG activation is reliably evoked by exemplars from arbitrary stimulus categories that are mapped by experimental rules onto an arbitrary covert or overt response.

Segregation of working memory functions within the dorsolateral prefrontal cortex.

It is now widely accepted that the prefrontal cortex (PFC) plays a critical role in the neural network subserving working memory (WM). At least three related questions are still under debate: (1) is the PFC critical for all constituent processes of WM (i.e., short-term storage, manipulation, and utilization of mental representations) or only in one or a few of them? (2) Is there segregation of function among different cytoarchitectonic subdivisions of the PFC? (3) If this be the case, is this segregation based on the nature of the information being processed or on the type of cognitive operation performed? The present review article describes findings in the monkey supporting a modular "domain-specific" model of PFC functional organization with respect to WM operations. In this model, the dorsolateral prefrontal cortex (DLPFC) is composed of several subregions, based primarily on the nature of the information being processed in WM. Storage and processing functions are integrally related in each area. Future studies designed to map as yet uncharted areas of prefrontal cortex with refined anatomical and physiological approaches may provide a critical test of the model and evaluate the extent to which it applies generally or, instead, mainly to visual domains or only to dorsolateral convexity areas.

Prominence of direct entorhinal-CA1 pathway activation in sensorimotor and cognitive tasks revealed by 2-DG functional mapping in nonhuman primate.

The trisynaptic pathway from entorhinal cortex to the hippocampus has long been regarded as the major route of information transfer underlying memory consolidation. Most physiological studies of this pathway involve recording from hippocampal slices. We have used both single- and double-label 2-deoxyglucose autoradiographic methods to image the pattern of activation in the hippocampal formation of 14 rhesus monkeys performing cognitive tasks, varying in content (spatial or nonspatial), process (working memory or associative memory), and mode of response (oculomotor or manual). These studies revealed a highly differentiated pattern of metabolic activation throughout the rostrocaudal extent of the hippocampal formation that was common to all behavioral conditions examined. This pattern consisted of intense activation of the stratum lacunosum-moleculare of CA1 and the subiculum, contrasting with barely detectable activity in CA3 and modest activation in the dentate gyrus, which did not include its molecular layer. These findings indicate a remarkable invariance in hippocampal activation under conditions of varied content, varied process, and varied mode of response and an heretofore-unappreciated preferential engagement of the direct rather than the trisynaptic pathway during performance of a wide range of behavioral tasks.