Subpopulation Of Cortical Neurons Research Articles

Investigating the interplay between segregation and integration in developing cortical assemblies.

The human brain is an intricate structure composed of interconnected modular networks, whose organization is known to balance the principles of segregation and integration, enabling rapid information exchange and the generation of coherent brain states. Segregation involves the specialization of brain regions for specific tasks, while integration facilitates communication among these regions, allowing for efficient information flow. Several factors influence this balance, including maturation, aging, and the insurgence of neurological disorders like epilepsy, stroke, or cancer. To gain insights into information processing and connectivity recovery, we devised a controllable in vitro model to mimic and investigate the effects of different segregation and integration ratios over time. We designed a cross-shaped polymeric mask to initially establish four independent sub-populations of cortical neurons and analyzed how the timing of its removal affected network development. We evaluated the morphological and functional features of the networks from 11 to 18 days in vitro (DIVs) with immunofluorescence techniques and micro-electrode arrays (MEAs). The removal of the mask at different developmental stages of the network lead to strong variations in the degree of intercommunication among the four assemblies (altering the segregation/integration balance), impacting firing and bursting parameters. Early removal (after 5 DIVs) resulted in networks with a level of integration similar to homogeneous controls (without physical constraints). In contrast, late removal (after 15 DIVs) hindered the formation of strong inter-compartment connectivity, leading to more clustered and segregated assemblies. A critical balance between segregation and integration was observed when the mask was removed at DIV 10, allowing for the formation of a strong connectivity among the still-separated compartments, thus demonstrating the existence of a time window in network development in which it is possible to achieve a balance between segregation and integration.

Distinct neuronal types contribute to hybrid temporal encoding strategies in primate auditory cortex.

Studies of the encoding of sensory stimuli by the brain often consider recorded neurons as a pool of identical units. Here, we report divergence in stimulus-encoding properties between subpopulations of cortical neurons that are classified based on spike timing and waveform features. Neurons in auditory cortex of the awake marmoset (Callithrix jacchus) encode temporal information with either stimulus-synchronized or nonsynchronized responses. When we classified single-unit recordings using either a criteria-based or an unsupervised classification method into regular-spiking, fast-spiking, and bursting units, a subset of intrinsically bursting neurons formed the most highly synchronized group, with strong phase-locking to sinusoidal amplitude modulation (SAM) that extended well above 20 Hz. In contrast with other unit types, these bursting neurons fired primarily on the rising phase of SAM or the onset of unmodulated stimuli, and preferred rapid stimulus onset rates. Such differentiating behavior has been previously reported in bursting neuron models and may reflect specializations for detection of acoustic edges. These units responded to natural stimuli (vocalizations) with brief and precise spiking at particular time points that could be decoded with high temporal stringency. Regular-spiking units better reflected the shape of slow modulations and responded more selectively to vocalizations with overall firing rate increases. Population decoding using time-binned neural activity found that decoding behavior differed substantially between regular-spiking and bursting units. A relatively small pool of bursting units was sufficient to identify the stimulus with high accuracy in a manner that relied on the temporal pattern of responses. These unit type differences may contribute to parallel and complementary neural codes.

Multi-Omic Analyses of Growth Cones at Different Developmental Stages Provides Insight into Pathways in Adult Neuroregeneration.

SummaryGrowth cones (GCs) are structures associated with growing neurons. GC membrane expansion, which necessitates protein-lipid interactions, is critical to axonal elongation in development and in adult neuritogenesis. We present a multi-omic analysis that integrates proteomics and lipidomics data for the identification of GC pathways, cell phenotypes, and lipid-protein interactions, with an analytic platform to facilitate the visualization of these data. We combine lipidomic data from GC and adult axonal regeneration following optic nerve crush. Our results reveal significant molecular variability in GCs across developmental ages that aligns with the upregulation and downregulation of lipid metabolic processes and correlates with distinct changes in the lipid composition of GC plasmalemma. We find that these processes also define the transition into a growth-permissive state in the adult central nervous system. The insight derived from these analyses will aid in promoting adult regeneration and functional innervation in devastating neurodegenerative diseases.

Stereotypic wheel running decreases cortical activity in mice.

Prolonged wakefulness is thought to gradually increase ‘sleep need' and influence subsequent sleep duration and intensity, but the role of specific waking behaviours remains unclear. Here we report the effect of voluntary wheel running during wakefulness on neuronal activity in the motor and somatosensory cortex in mice. We find that stereotypic wheel running is associated with a substantial reduction in firing rates among a large subpopulation of cortical neurons, especially at high speeds. Wheel running also has longer-term effects on spiking activity across periods of wakefulness. Specifically, cortical firing rates are significantly higher towards the end of a spontaneous prolonged waking period. However, this increase is abolished when wakefulness is dominated by running wheel activity. These findings indicate that wake-related changes in firing rates are determined not only by wake duration, but also by specific waking behaviours.

Glia: Anchored at the axon.

A subset of perineuronal satellite microglia interact specifically with the axon initial segment in a subpopulation of cortical neurons.

Behavioural neuroscience: socially interactive interneurons.

A subpopulation of cortical neurons that express oxytocin receptors mediates female sociosexual behaviour in mice.

Reliable in vivo identification of both GABAergic and glutamatergic neurons using Emx1-Cre driven fluorescent reporter expression

The development of genetically modified mice in which subpopulations of cortical neurons are labelled by fluorescent proteins has greatly facilitated single-cellular imaging and electrophysiology studies in vitro and in vivo. However, the parallel visualization of both inhibitory and excitatory neocortical neurons remains problematic. We here provide an alternative approach to identify GABAergic neurons in the context of in vivo calcium imaging. The method relies on the Emx1IREScre recombinase driven expression of a red fluorescent protein in excitatory neurons and glia. We quantitatively examined the upper layers of the visual neocortex in vivo and found that due to pronounced neuropil staining Emx1IREScre-negative and Emx1IREScre-positive neurons can be reliably differentiated based on negative and positive contrast, respectively. Immunohistochemical analyses confirmed that the entire population of GABAergic interneurons is represented by Emx1IREScre-negative cells. The potential usefulness of the method is exemplified by calcium imaging of sensory-evoked responses in the primary visual cortex. We conclude that the proposed method extends the repertoire of strategies aimed at discriminating two major neocortical neuron populations in situ.

A Mammalian Conserved Element Derived from SINE Displays Enhancer Properties Recapitulating Satb2 Expression in Early-Born Callosal Projection Neurons

Short interspersed repetitive elements (SINEs) are highly repeated sequences that account for a significant proportion of many eukaryotic genomes and are usually considered “junk DNA”. However, we previously discovered that many AmnSINE1 loci are evolutionarily conserved across mammalian genomes, suggesting that they may have acquired significant functions involved in controlling mammalian-specific traits. Notably, we identified the AS021 SINE locus, located 390 kbp upstream of Satb2. Using transgenic mice, we showed that this SINE displays specific enhancer activity in the developing cerebral cortex. The transcription factor Satb2 is expressed by cortical neurons extending axons through the corpus callosum and is a determinant of callosal versus subcortical projection. Mouse mutants reveal a crucial function for Sabt2 in corpus callosum formation. In this study, we compared the enhancer activity of the AS021 locus with Satb2 expression during telencephalic development in the mouse. First, we showed that the AS021 enhancer is specifically activated in early-born Satb2+ neurons. Second, we demonstrated that the activity of the AS021 enhancer recapitulates the expression of Satb2 at later embryonic and postnatal stages in deep-layer but not superficial-layer neurons, suggesting the possibility that the expression of Satb2 in these two subpopulations of cortical neurons is under genetically distinct transcriptional control. Third, we showed that the AS021 enhancer is activated in neurons projecting through the corpus callosum, as described for Satb2+ neurons. Notably, AS021 drives specific expression in axons crossing through the ventral (TAG1−/NPY+) portion of the corpus callosum, confirming that it is active in a subpopulation of callosal neurons. These data suggest that exaptation of the AS021 SINE locus might be involved in enhancement of Satb2 expression, leading to the establishment of interhemispheric communication via the corpus callosum, a eutherian-specific brain structure.

High frequency of development of B cell lymphoproliferation and diffuse large B cell lymphoma in Dbl knock-in mice

Dbl is the prototype of a large family of GDP-GTP exchange factors for small GTPases of the Rho family. In vitro, Dbl is known to activate Rho, Rac, and Cdc42 and to induce a transformed phenotype in murine fibroblasts. We previously reported that Dbl-null mice are viable and fertile but display defective dendrite elongation of distinct subpopulations of cortical neurons, suggesting a role of Dbl in controlling dendritic growth. To gain deeper insights into the role of Dbl in development and disease, we attempted a knock-in approach to create an endogenous allele that encodes a missense-mutation-mediated loss of function in the DH domain. We generated, by gene targeting technology, a mutant mouse strain by inserting a mutagenized human proto-Dbl cDNA clone expressing only the Dbl N terminus regulatory sequence at the starting codon of murine exon 1. Animals were monitored over a 21-month period, and necropsy specimens were collected for histological examination and immunohistochemistry analysis. Dbl knock-in mice are viable and did not manifest either decreased reproductive performances or gross developmental phenotype but revealed a reduced lifespan compared to wild-type (w.t.) mice and showed, with aging, a B cell lymphoproliferation that often has features of a frank diffuse large B cell lymphoma. Moreover, Dbl knock-in male mice displayed an increased incidence of lung adenoma compared to w.t. mice. These data indicate that Dbl is a tumor susceptibility gene in mice and that loss of function of Dbl DH domain by genetic missense mutations is responsible for induction of diffuse large B cell lymphoma.

PINP: A New Method of Tagging Neuronal Populations for Identification during In Vivo Electrophysiological Recording

Neural circuits are exquisitely organized, consisting of many different neuronal subpopulations. However, it is difficult to assess the functional roles of these subpopulations using conventional extracellular recording techniques because these techniques do not easily distinguish spikes from different neuronal populations. To overcome this limitation, we have developed PINP (Photostimulation-assisted Identification of Neuronal Populations), a method of tagging neuronal populations for identification during in vivo electrophysiological recording. The method is based on expressing the light-activated channel channelrhodopsin-2 (ChR2) to restricted neuronal subpopulations. ChR2-tagged neurons can be detected electrophysiologically in vivo since illumination of these neurons with a brief flash of blue light triggers a short latency reliable action potential. We demonstrate the feasibility of this technique by expressing ChR2 in distinct populations of cortical neurons using two different strategies. First, we labeled a subpopulation of cortical neurons—mainly fast-spiking interneurons—by using adeno-associated virus (AAV) to deliver ChR2 in a transgenic mouse line in which the expression of Cre recombinase was driven by the parvalbumin promoter. Second, we labeled subpopulations of excitatory neurons in the rat auditory cortex with ChR2 based on projection target by using herpes simplex virus 1 (HSV1), which is efficiently taken up by axons and transported retrogradely; we find that this latter population responds to acoustic stimulation differently from unlabeled neurons. Tagging neurons is a novel application of ChR2, used in this case to monitor activity instead of manipulating it. PINP can be readily extended to other populations of genetically identifiable neurons, and will provide a useful method for probing the functional role of different neuronal populations in vivo.

Expression of apoptosis-inducing factor (AIF) in the aged rat brain

The mitochondrial flavoprotein apoptosis-inducing factor (AIF) promotes cell death upon nuclear translocation or by impinging on mitochondrial respiratory complex-I activity. Because decreased complex-I activity is associated with brain aging, we investigated the expression and distribution of AIF in frontal cortex, hippocampus and striatum of aged Long-Evans rat brains. We found that AIF was: (i) more abundantly expressed in striatum than in the other two brain regions, (ii) enriched in deep layers of frontal cortex and in the pyramidal cell layer of hippocampus, and (iii) overall mainly localized to mitochondria, but significantly more translocated to the nucleus in the deep layers of frontal cortex. Altogether, our data point to a difference in region 1 AIF expression patterns, and provide evidence for the involvement of AIF in the cell death of a subpopulation of cortical neurons in aged animals.

Progestin receptor is transiently expressed perinatally in neurons of the rat isocortex

Steroid hormones influence the development of numerous brain regions, including some that are not classically considered steroid-sensitive. For example, nuclear receptors for both androgen and estrogen have been detected in neonatal cortical cells. High levels of progestin binding and progestin receptor (PR) mRNA have also been reported in early perinatal isocortex. PR expression coincides with high levels of de novo progesterone produced within the cortex, suggesting that PR and its ligand influence the important developmental cortical processes occurring shortly after birth. In order to better understand the role PR plays in cortical development, we used the cellular-level resolution of immunohistochemistry and in situ hybridization (ISH) to characterize changes in perinatal PR expression within specific cortical lamina. PR immunoreactivity (PR-ir) was examined at embryonic days (E) 18, 20, 21, 22, and postnatal days (P) 1, 3, 6, 9, 13, and 27. We find that PR-ir is transiently expressed in specific lamina of frontal, parietal, temporal, and occipital cortex. PR-ir was observed in subplate cells on E18, in increasingly superficial lamina (primarily lamina V, then II/III) during early postnatal development, and was absent by P27. Double-labeling immunohistochemistry indicated that PR-ir colocalizes with the neuronal marker, microtubule associated protein-2, but not with the glial marker, nestin, nor with gamma-aminobutyric acid. These results suggest that specific subpopulations of cortical neurons may be transiently sensitive to progesterone, and that progesterone and its receptor may play a critical role in the fundamental mechanisms underlying normal cortical development.

Simultaneous recordings of visual evoked potentials and BOLD MRI activations in response to visual motion processing

Visual motion processing in humans was studied by simultaneous 32-channel electroencephalography (EEG) recordings of visual evoked potentials and BOLD MRI activations at 2.9 T. The paradigms compared three different random dot patterns (12 s duration) with stationary random dots (18 s) or with each other. The stimuli represented pattern reversal (500 ms switches between two stationary patterns), motion onset (200 ms of starfield motion followed by 1000 ms of stationary dots) and motion reversal (reversal of moving starfield directions every 1000 ms). Whereas motion-evoked visual potentials, and in particular the N2 component in occipito-temporal channels, were most prominent for motion onset, the most extended BOLD MRI activations and strongest signal changes in V5/MT+ were obtained in response to motion reversal. These apparently contradictory findings most likely reflect different physiological aspects of the neural activity associated with visual motion processing. For example, desynchronized activity of subpopulations of cortical neurons inside V5/MT+ is expected to attenuate visual evoked potentials in scalp recordings while continuously driving metabolic demands that lead to sustained BOLD MRI responses. The understanding of the physiological correlates and neural processes underlying either technique is fundamental to exploring fully the potential of combined EEG-MRI for studying human brain function at both high temporal and spatial resolution.

A microelectrode array (MEA) integrated with clustering structures for investigating in vitro neurodynamics in confined interconnected sub-populations of neurons

Understanding how the information is coded in large neuronal networks is one of the major challenges for neuroscience. A possible approach to investigate the information processing capabilities of the neuronal ensembles is given by the use of dissociated neuronal cultures coupled to microelectrode arrays (MEAs). Here, we describe a new strategy, based on MEAs, for studying in vitro neuronal network dynamics in interconnected sub-populations of cortical neurons. The rationale is to sub-divide the neuronal network into communicating clusters while preserving a high degree of functional connectivity within each confined sub-population, i.e. to achieve a compromise between a completely random large neuronal population and a patterned network, such as currently used with conventional MEAs. To this end, we have realized and functionally characterized a Pt microelectrode array with an integrated EPON SU-8 clustering structure, allowing to confine five relatively large yet interconnected spontaneously developing neuronal networks (i.e. thousands of cells). The clustering structure consists of five chambers of 3 mm in diameter interconnected via 800 μm long and 300 μm wide microchannels and is integrated on the MEA of 60 thin-film Pt electrodes of 30 μm diameter. Tests of the Pt microelectrodes’ stability under stimulation showed a stable behavior up to 35,000 voltage stimuli and the biocompatibility was assessed with the cultures of dissociated rat's cortical neurons achieving cultures’ viability up to 60 days in vitro. Compared to conventional MEAs, the monitoring of spontaneous and evoked activity and the computation of the Post-Stimulus Time Histogram (PSTH) within the clusters clearly demonstrates: (i) the capability to selectively activate (through poly-synaptic pathways) specific network regions and (ii) the confinement of the network dynamics mainly in the highly connected sub-networks.

Hyperexcitability of the primary somatosensory cortex in migraine--a magnetoencephalographic study.

The excitability of the cerebral cortex in the interictal state of migraine appears to be fundamental in the brain's susceptibility to migraine attacks. Subpopulations of cortical neurons are reported to have different physiological response properties to different interstimulus intervals (ISIs) and, hence, may be differentially altered or modulated in migraine. The aim of this study therefore was to evaluate response characteristics of temporally and spatially defined neuronal subpopulations in the cortex of migraineurs. To this end, we measured, by means of magnetoencephalography (37-channel neuromagnetometer), the response properties of the early components of the somatosensory evoked magnetic fields following electrical stimulation of the median nerve, the N20m and P35m, at ISIs ranging between 0.3 and 6 s. As a measure of the number of excited neurons underlying the N20m and P35m, we evaluated the root mean square (r.m.s.) of the deflections across all 37 channels at the corresponding latencies and the corresponding dipole moment of the equivalent current dipole (ECD strength). Twenty consecutive women with at least three migraine attacks/month (range 3-8/month) fulfilling the International Headache Society criteria and 20 age-matched healthy women were included in the study. In migraineurs, the r.m.s. and ECD strength of N20m was increased at all ISIs (r.m.s., P < 0.05; ECD strength, P < 0.01) and positively related to the mean attack frequency (r.m.s., R(s) = 0.6, P < 0.01; ECD strength, R(s) = 0.5, P < 0.05). In contrast, the r.m.s. and ECD strength of P35m did not differ significantly between migraineurs and control subjects and did not correlate significantly with the frequency of migraine attacks. Responses to different ISIs did not differ significantly between migraineurs and control subjects. The r.m.s. of N20m was stable for ISIs between 0.5 and 6 s and decreased significantly at an ISI of 0.3 s. In contrast, the r.m.s. of P35m decreased continuously as the ISI was decreased below 6 s and this reached significance for an ISI of < or =1 s. Habituation of N20m or P35m, i.e. a decrease in response magnitude following repetitive stimulation over time, was not found in either the control subjects or in the migraineurs. It is concluded that the population of neurons in the primary somatosensory cortex underlying the N20m are hyperexcitable and that this hyperexcitability is linked to the frequency of migraine attacks. This hyperexcitability appears not to be related to habituation since habituation was not found in the control subjects. In contrast, the magnitude of P35m is not pathophysiologically linked to the interictal state of migraine. Furthermore, the cellular mechanisms causing ISI-dependent depression of N20m and P35m are not altered in migraine.

Subcellular localization of alpha-synuclein in primary neuronal cultures: effect of missense mutations.

Numerous recent observations have implicated alpha-synuclein in the pathogenesis of several neurodegenerative diseases, including Parkinson's disease, Alzheimer's disease, dementia with Lewy bodies and multiple-system atrophy. Two missense mutations in the gene for alpha-synuclein have been identified in some cases of familial Parkinson's disease and it is thought that these may disrupt the normal structure of the protein and thus promote aggregation into Lewy body filaments. Here, we examine the subcellular localization of alpha-synuclein in primary cortical neurons maintained in a monolayer culture. The protein has widespread expression throughout neurons, including the nucleus, and has a discete localization in the neurites of more mature neurons, reminiscent of synaptic specializations. Interestingly, in a subpopulation of cortical neurons transfected at 13 days in vitro, we find that alpha-synuclein appears to aggregate into distinct punctate inclusions in the cytoplasm and proximal neurites. Unlike Lewy bodies, these structures are not ubiquitin positive. These regions of alpha-synuclein accumulation are observed following transfections with wild-type, Ala30Pro or Ala53Thr alpha-synuclein; neither mutation alters their frequency.

Quantification of Fos immunoreactivity in cortical cultures treated with growth factors

Among the effects mediated by growth factors in vivo are the switch to growth arrest and differentiation during normal development of the nervous system, survival during the period of naturally occurring cell death, and plasticity and repair of neurons in the adult brain. Much interest has focused on the signalling pathways utilised by growth factors with a large proportion of experiments carried out using the phaeochromocytoma cell line. Here we have quantified Fos immunoreactivity following stimulation of primary cultures of rat cortical neurons with a variety of growth factors including neurotrophins and cytokines. Expression of Fos has been quantified in these cultures using an ELISA technique, and immunocytochemistry followed by digital stereology. Treatment of cultures with brain derived neurotrophic factor (BDNF) or neurotrophin-4 (NT-4) causes a dose-dependent increase in Fos expression, while neurotrophin-3 (NT-3) causes an increase but at high concentrations only. A sub-population of cortical neurons within the cultures express Fos in response to fibroblast growth factor-1 or fibroblast growth factor-2 but no cells respond with Fos expression on treatment with insulin-like growth factor-I. We conclude that BDNF and NT-4 cause dose-dependent increases in the number of Fos immunoreactive cells.

Kainate-stimulated Zn 2+ uptake labels cortical neurons with Ca 2+-permeable AMPA/kainate channels

The endogenous cation, Zn 2+, is synaptically released and may trigger neurodegeneration after permeating through NMDA channels, voltage sensitive Ca 2+ channels (VSCC), or Ca 2+ permeable AMPA/kainate channels (Ca-A/K). Neurons expressing Ca-A/K can be identified by a histochemical stain based upon kainate-stimulated Co 2+ uptake (Co 2+(+) neurons). The primary objective of this study was to determine whether a similar approach could be employed to visualize agonist-stimulated intracellular Zn 2+ accumulation, and, thus, to test the hypothesis that Ca-A/K permit particularly rapid Zn 2+ flux. Substituting Zn 2+ for Co 2+ during agonist-stimulated uptake, followed by Timm's sulfide–silver staining to visualize intracellular Zn 2+, resulted in distinct labeling of a subpopulation of cortical neurons (Zn 2+(+) neurons) closely resembling Co 2+(+) neurons, suggesting that, like Co 2+, Zn 2+ may permeate Ca-A/K with particular rapidity. Neither NMDA nor high K + triggered comparable Zn 2+ accumulation, indicating substantially greater permeation through Ca-A/K than through NMDA channels or VSCC. Both fluorescence studies of intracellular Zn 2+ accumulation and double staining studies (using SMI-32 and anti-glutamate decarboxylase antibodies, both markers of cortical neuronal subsets), support the contention that Zn 2+ and Co 2+ labeling identify a common set of neurons characterized by expression of AMPA/kainate channels directly permeable to Zn 2+ and Co 2+ as well as Ca 2+. Furthermore, the preferential destruction of Zn 2+(+) neurons (like Co 2+(+) neurons) after brief kainate exposures in the presence of lower, more physiologic concentrations of Zn 2+ suggests that Zn 2+ permeation through Ca-A/K could contribute to selective neurodegeneration in disease. Finally, the study provides a novel and potentially advantageous histochemical approach for kainate-stimulated Co 2+ or Zn 2+ uptake labeling, using a room temperature technique (Timm's staining) rather than the usual hot AgNO 3 development of the Co 2+ stain.

Synchronous development of pyramidal neuron dendritic spines and parvalbumin-immunoreactive chandelier neuron axon terminals in layer III of monkey prefrontal cortex

Postnatal development of the primate cerebral cortex involves an initial proliferation and the subsequent attrition of cortical synapses. Although these maturational changes in synaptic density have been observed across the cortical mantle, little is known about the precise time course of developmental refinements in synaptic inputs to specific populations of cortical neurons. We examined the postnatal development of two markers of excitatory and inhibitory inputs to a subpopulation of layer III pyramidal neurons in area 9 and 46 of rhesus monkey prefrontal cortex. These neurons are of particular interest because they play a major role in the flow of information both within and between cortical regions. Quantitative reconstructions of Golgi-impregnated mid-layer III pyramidal neurons revealed substantial developmental changes in the relative density of dendritic spines, the major site of excitatory inputs to these neurons. Relative spine density on both the apical and basilar dendritic trees increased by 50% during the first two postnatal months, remained at a plateau through 1.5 years of age, and then decreased over the peripubertal age range until stable adult levels were achieved. As a measure of the postnatal changes in inhibitory input to the axon initial segment of these pyramidal neurons, we determined the density of parvalbumin-immunoreactive axon terminals belonging to the chandelier class of local circuit neurons. The density of these distinctive axon terminals (cartridges) exhibited a temporal pattern of change that exactly paralleled the changes in dendritic spine density. These results suggest that subpopulations of cortical neurons may be regulated by dynamic interactions between excitatory and inhibitory inputs during development and, in concert with other data, they emphasize the cellular specificity of postnatal refinements in cortical circuitry.

Cellular colocalization of dopamine D1 and D2 receptors in rat medial prefrontal cortex.

In a recent study in rat medial prefrontal cortex (mPFC), a fluorescently coupled, high-affinity ligand for the D1 receptor subtype was localized to nonpyramidal neurons, while a ligand selective for the D2 subtype was found on neurons with a size distribution overlapping with both small pyramidal and large nonpyramidal cells. These observations raised the possibility that a subpopulation of cortical neurons with an intermediate size range may coexpress both the D1 and D2 receptor subtypes. In the present study, the D1 and D2 receptor subtypes have been simultaneously localized in layer VI of rat mPFC using 20 nM SCH 23390-Bodipy and 20 nM N-(p-aminophenethyl) spiperone-Texas red, respectively, in the presence of 100 nM mianserin (5-HT2 receptor antagonist). The localization of receptor binding fluorescence was assessed in paired images using fluoroscein isothiocyanate (FITC) and rhodamine dichroic filters for the D1 and D2 subtypes, respectively. Under the conditions employed here, most cell bodies showed either D1-like or D2-like receptor binding fluorescence, while a colocalization of both fluoroprobes was observed on only 25% of the labeled cells. When the size of each single-labeled cell body was measured using the respective FITC (D1-probe) and rhodamine (D2-probe) epifluorescence filters, the distribution of cells showing only D1-like receptor binding fluorescence was similar to nonpyramidal neurons (68.6 +/- 1.8 microns 2), while that for cells showing only D2-like receptor binding fluorescence was similar to that of both large interneurons and small pyramidal cells (106.9 +/- 2.4 microns 2).(ABSTRACT TRUNCATED AT 250 WORDS)