Laminar and Cellular Distribution of Monoamine Receptors in Rat Medial Prefrontal Cortex

Abstract

The prefrontal cortex (PFC) is deeply involved in higher brain functions, many of which are altered in psychiatric conditions. The PFC exerts a top-down control of most cortical and subcortical areas through descending pathways and is densely innervated by axons emerging from the brainstem monoamine cell groups, namely, the dorsal and median raphe nuclei (DR and MnR, respectively), the ventral tegmental area and the locus coeruleus (LC). In turn, the activity of these cell groups is tightly controlled by afferent pathways arising from layer V PFC pyramidal neurons. The reciprocal connectivity between PFC and monoamine cell groups is of interest to study the pathophysiology and treatment of severe psychiatric disorders, such as major depression and schizophrenia, inasmuch as antidepressant and antipsychotic drugs target monoamine receptors/transporters expressed in these areas. Here we review previous reports examining the presence of monoamine receptors in pyramidal and GABAergic neurons of the PFC using double in situ hybridization. Additionally, we present new data on the quantitative layer distribution (layers I, II-III, V, and VI) of monoamine receptor-expressing cells in the cingulate (Cg), prelimbic (PrL) and infralimbic (IL) subfields of the medial PFC (mPFC). The receptors examined include serotonin 5-HT_1A, 5-HT_2A, 5-HT_2C, and 5-HT₃, dopamine D₁ and D₂ receptors, and α_1A-, α_1B-, and α_1D-adrenoceptors. With the exception of 5-HT₃ receptors, selectively expressed by layers I-III GABA interneurons, the rest of monoamine receptors are widely expressed by pyramidal and GABAergic neurons in intermediate and deep layers of mPFC (5-HT_2C receptors are also expressed in layer I). This complex distribution suggests that monoamines may modulate the communications between PFC and cortical/subcortical areas through the activation of receptors expressed by neurons in intermediate (e.g., 5-HT_1A, 5-HT_2A, α_1D-adrenoceptors, dopamine D₁ receptors) and deep layers (e.g., 5-HT_1A, 5-HT_2A, α_1A-adrenoceptors, dopamine D₂ receptors), respectively. Overall, these data provide a detailed framework to better understand the role of monoamines in the processing of cognitive and emotional signals by the PFC. Likewise, they may be helpful to characterize brain circuits relevant for the therapeutic action of antidepressant and antipsychotic drugs and to improve their therapeutic action, overcoming the limitations of current drugs.

Keywords: 5-hydroxytryptamine (serotonin) receptors; antidepressant drugs; antipsychotic drugs; cortical layers; dopamine receptors; major depressive disorder; noradrenaline receptors; schizophrenia.

FIGURE 1

High magnification photomicrographs showing the presence of 5-HT_1A receptor mRNA (³³P-labeled oligonucleotides) in pyramidal cells, identified by the presence of VGLUT1 mRNA (Dig-labeled oligonucleotides). Both images were acquired from the same experiment and correspond to deep layers of mPFC cingulate area. (A) Was captured in 2003 with a Nikon Eclipse E1000 microscope (Nikon, Tokyo, Japan) using a digital camera (DXM1200 3.0; Nikon) and analySIS Software (Soft Imaging System GmbH, Germany); (B) was captured in 2017 with a Zeiss Axioplan microscope equipped with a digital camera (XC50, Olympus) with Olympus CellSens Entry software.

FIGURE 2

mRNA expression of monoaminergic receptors in rat prefrontal cortex (PFC). (A1) Coronal diagram from the rat brain atlas Swanson (2004) (used under CC BY-NC 4.0) at the approximate AP coordinate where cell counts have been performed in the cingulate (Cg), prelimbic (PrL), and infralimbic (IL) subdivisions. (A2–A10) Emulsion dipped dark-field PFC coronal sections hybridized with ³³P-labeled oligonucleotide probes against the mRNAs encoding 5-HT_1A-R (A2), 5-HT_2A-R (A3), 5-HT_2C-R (A4), 5-HT₃-R (A5) dopamine D₁-R (A6), dopamine D₂-R (A7) and α_1A-, α_1B- and α_1D-adrenoceptors (A8–A10, respectively). Bar: 1 mm. See Puig et al. (2004), Santana et al. (2004, 2009, 2013), Santana and Artigas (2017) for detailed methods.

FIGURE 3

Bar graphs showing the expression of each monoamine receptor mRNA in the different layers and PFC subfields of the mPFC. Data (mean of 3 rats, 2–3 consecutive sections per rat) are the percentages of pyramidal (VGLUT1-positive) or GABAergic (GAD-positive) neurons expressing each mRNA, expressed as percentages of the total neuronal population, assuming a standard 80% of pyramidal neurons and 20% of GABAergic interneurons. As layer I lacks glutamatergic neurons, layer I data is expressed as percentages of GAD-positive cells containing each receptor mRNA. Cg, anterior cingulate cortex; PL, prelimbic cortex; IL infralimbic cortex. Layer I, II–III, V, and VI stand for medial prefrontal cortical layers I to VI, respectively.

FIGURE 4

Percentages of pyramidal (VGLUT1-positive) and GABA (GAD-positive) neurons expressing the nine monoamine receptors studied in the different layers of cingulate area of medial prefrontal cortex. ^ap < 0.05 vs. rest of layers, ^a′p < 0.05 vs. layer I; ^a″′p < 0.05 vs. layer II–III; ^a″′p < 0.05 vs. layer V (for VGLUT1 graphs). ^bp < 0.05 vs. rest of layers, ^b′p < 0.05 vs. layer I; ^b″p < 0.05 vs. layers II–III; ^b″′p < 0.05 vs. layer V (for GAD graphs), one-way ANOVA followed by Tukey’s test.

FIGURE 5

Percentages of pyramidal (VGLUT1-positive) and GABA (GAD-positive) neurons expressing the nine monoamine receptors studied in the different layers of prelimbic area of medial prefrontal cortex. ^ap < 0.05 vs. rest of layers, ^a′p < 0.05 vs. layer I; ^a″p < 0.05 vs. layers II–III; ^a″′p < 0.05 vs. layer V (for VGLUT1 graphs). ^bp < 0.05 vs. rest of layers, ^b′p < 0.05 vs. layer I; ^b″p < 0.05 vs. layers II–III; ^b″′p < 0.05 vs. layer V (for GAD graphs), one-way ANOVA followed by Tukey’s test.

FIGURE 6

Percentages of pyramidal (VGLUT1-positive) and GABA (GAD-positive) neurons expressing the nine monoamine receptors studied in the different layers of infralimbic area of medial prefrontal cortex. ^ap < 0.05 vs. rest of layers, ^a′p < 0.05 vs. layer I; ^a″p < 0.05 vs. layers II–III; ^a″′p < 0.05 vs. layer V (for VGLUT1 graphs). ^bp < 0.05 vs. rest of layers, ^b′p < 0.05 vs. layer I; ^b″p < 0.05 vs. layers II–III; ^b″′p < 0.05 vs. layer V (for GAD graphs), one-way ANOVA followed by Tukey’s test.

FIGURE 7

(A,B) are peristimulus time histograms showing the orthodromic excitations elicited by the electrical stimulation of the DR at a physiological rate (0.5–1.7 mA, 0.2 ms square pulses, 0.9 Hz) on (A) a putatively GABAergic, 5-HT₃-R-expressing neuron and (B) on a layer V pyramidal neuron in the prelimbic PFC, identified by antidromic stimulation from midbrain (note the antidromic potential, arrowhead). Both responses were selectively blocked by the administration of the respective antagonists ondansetron (A) and M100907 (B) (not shown). The 5-HT₃-R-mediated responses in putative GABAergic neurons were faster and more effective than those evoked by 5-HT_2A-R activation in pyramidal neurons, due to the ionic nature of 5-HT₃-R. The concordance rates of the units shown are 85% (A) and 43% (B), i.e., 100 electric stimuli delivered in the DR evoked 80 action potentials in GABAergic interneurons through 5-HT₃-R activation, compared to 45 action potentials evoked in layer V pyramidal neurons, mediated by the activation of 5-HT_2A-R. Each peristimulus consists of 200 triggers; bin size is 4 ms. The arrow at zero abscissa marks the stimulation artifact. (C) Composite photomicrographs showing the localization of cells expressing VGLUT1, GAD, 5-HT₃-R, and 5-HT_2A–R mRNAs through layers I–VI at the level of the prelimbic PFC. The continuous vertical line denotes the location of the midline whereas the dotted line shows the approximate border between layers I and II. Pyramidal neurons (as visualized by VGLUT1 mRNA) are present in layers II–VI whereas GAD mRNA-positive cells are present in all layers, including layer I. Note the different location of cells expressing 5-HT₃-R and 5-HT_2A–R. 5-HT₃-R transcript is expressed by a limited number of GABA interneurons in layers I–III, particularly in the border between layers I and II. However, they represent 40% of GABAergic neurons in layer I. Scale bar = 150 μm. Redrawn from Puig et al. (2004), by permission of Oxford University Press.