Serotonin2C receptor localization in GABA neurons of the rat medial prefrontal cortex: implications for understanding the neurobiology of addiction - PubMed (original) (raw)

Serotonin2C receptor localization in GABA neurons of the rat medial prefrontal cortex: implications for understanding the neurobiology of addiction

S Liu et al. Neuroscience. 2007.

Abstract

Serotonin (5-HT) action via the 5-HT(2C) receptor (5-HT(2C)R) provides an important modulatory influence over neurons of the prefrontal cortex (PFC), which is critically involved in disorders of executive function including substance use disorders. In the present study, we investigated the distribution of the 5-HT(2C)R in the rat prelimbic prefrontal cortex (PrL), a subregion of the medial prefrontal cortex (mPFC), using a polyclonal antibody raised against the 5-HT(2C)R. The expression of 5-HT(2C)R immunoreactivity (IR) was highest in the deep layers (layers V/VI) of the mPFC. The 5-HT(2C)R-IR was typically most intense at the periphery of cell bodies and the initial segment of cell processes. Approximately 50% of the 5-HT(2C)R-IR detected was found in glutamate decarboxylase, isoform 67 (GAD 67)-positive neurons. Of the subtypes of GABA interneurons identified by expression of several calcium-binding proteins, a significantly higher percentage of neurons expressing IR for parvalbumin also expressed 5-HT(2C)R-IR than did the percentage of neurons expressing calbindin-IR or calretinin-IR that also expressed 5-HT(2C)R-IR. Since parvalbumin is located in basket and chandelier GABA interneurons which project to cell body and initial axon segments of pyramidal cells, respectively, these results raise the possibility that the 5-HT(2C)R in the mPFC acts via the parvalbumin-positive GABAergic interneurons to regulate the output of pyramidal cells in the rat mPFC.

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Figures

Figure 1. Western blot analysis demonstrating detection of the 5-HT2CR in the PSD of the PFC

Total homogenate (Total) and PSD-associated proteins (PSD) from the PFC were analyzed by Western blot using antibodies against PSD-95 (a post-synaptic density marker), syntaxin (a pre-synaptic marker) and the 5-HT2CR. In addition, peptide neutralization of the 5-HT2CR antibody with its appropriate blocking peptide eliminated the anti-5-HT2CR antibody-associated band. Data presented are from a single blot that was stripped and re-probed. Location of the molecular weight markers are shown to the left of the blots.

Figure 2. Schematic drawing of representative section used for the quantification of IR cells in the PrL

The drawing (bregma 3.24 mm) was adapted from Paxinos and Watson (2004). The box shows the area in which photomicrographs at a magnification of 40x were taken to quantify the numbers of 5-HT2CR-IR cells in superficial (layers I and II/III) and deep layers (layers V and VI) of the PrL and their colocalization with GAD 67-, parvalbumin-, calbindin- or calretinin-IR. ACC: anterior cingulate cortex; PrL: prelimbic cortex; IL: infralimbic cortex.

Figure 3. Stratification and segregation of immunoreactive profiles for the 5-HT2CR-IR in the PrL

Expression of 5-HT2CR-IR (green, A) was segregated into specific cortical layers shown by comparison with neighboring sections (B) stained with cresyl violet**,** to aid in identifying the cortical layers (I–VI). Note that most 5-HT2CR-IR cells are located in layer V–VI. A few cells are also stained in layers I and II/III. (C) is a schematic drawing adapted from Paxinos and Watson (2004) corresponding to coronal sections (Bregma 3.24) from which images in A and B were captured. Scale bar: 200 μm. ACC: anterior cingulate cortex; PrL: prelimbic cortex; IL: infralimbic cortex.

Figure 4. Photomicrographs of immunostained neurons in the deep layers of the PrL illustrate the subcellular distribution patterns of IR associated with each of the immunoreagents used in this study

The 5-HT2CR-IR (green, A) was typically most intense at the periphery of the cell bodies, with some extension into the proximal dendrites; DAPI stained nuclei are also shown (blue). The antisera against GAD 67 (red, B) produced a relatively homogeneous staining of the cell soma and occasionally weakly labeled the proximal dendrites. The antisera against parvalbumin (red, C), calbindin (red, D) and calretinin (red, E) produced a diffuse staining that labeled the soma and proximal and distal neurites. Scale bar, 20 μm.

Figure 5. Colocalization of GAD 67 and 5-HT2CR in layer IV of the PrL

Photomicrographs of double-label immunofluorescent staining for the 5-HT2CR (green, A), and GAD 67 (red, B). Figure C displays the overlay of images in A and B to demonstrate colocalization. Approximately 50% of 5-HT2CR-positive cells in the field also contain GAD 67-IR. Examples of cells that express immunoreactivity for GAD 67 and 5-HT2CR are shown by arrows. D. Schematic drawing adapted from Paxinos and Watson (2004) corresponding to coronal sections (bregma 3.24 mm) from which images in A–C were captured. Scale bar: 50 μm. ACC: anterior cingulate cortex; PrL: prelimbic cortex; IL: infralimbic cortex.

Figure 6. Colocalization of calcium-binding proteins and 5-HT2CR in layer IV of the PrL

In all cases, the 5-HT2CR-IR is demonstrated by Alexa Fluor® 488 (green, A, D, G), and parvalbumin-, calbindin-, and calretinin are demonstrated by Alexa Fluor® 555 staining (red, B, E, H, respectively). Figures C, F and I display the overlay of images in A+B, D+E and G+H, respectively. Of the three classes of interneurons, double-labeled parvalbumin cells tended to express the greatest degree of 5-HT2CR-IR. Arrows show the cells with both parvalbumin-IR and 5-HT2CR-IR. All images were taken from the same area as depicted in Figure 5D. Scale bar: 100 μm.

Figure 7. Number of parvalbumin-, calbindin-, and calretinin-positive cells in the rat PrL

The average number of parvalbumin-, calbindin-, and calretinin-IR cells detected (± S.E.M.) in the PrL are presented (n = 3 rats). The number of calretinin- or calbindin-positive cells detected is significantly lower (p < 0.05) than the number of parvalbumin-positive cells detected in both the superficial layers (layers I–III; A) and the deep layers (layers V/VI; B) of the PrL.

Figure 8. Number of parvalbumin-, calbindin-, and calretinin-positive cells in the rat PrL that co-express 5-HT2CR-IR

Interneuron populations were defined by IR staining for parvalbumin, calbindin, or calretinin. The average of the percentage of total cells detected for each interneuron subpopulation that also contained 5-HT2CR-IR (± S.E.M.) is presented (n = 3 rats). The percentage of calretinin or calbindin-positive cells which express 5-HT2CR-IR is significantly lower (p < 0.05) than the number of parvalbumin-positive cells in the deep layers (layers V/VI; B) but not the superficial layers (layers I–III; A) of the PrL.

Figure 9. Schematic representation of the sites of action for the PrL 5-HT2CR to modulate PFC output to the VTA and NAc

The projection from 5-HT neurons in the raphe nuclei (Raphe) to the prefrontal cortex (PFC) is represented by the solid light blue line. The 5-HT2CR located in the PFC are represented by light blue hexagons. The GABA interneurons that express parvalbumin (GABA/PV; red), calbindin (GABA/CB; green) or calretinin (GABA/CR; yellow) are also illustrated. Pyramidal glutamate (Pyr) projections from the PFC to the VTA and NAc are represented by solid purple lines. Dopamine projections from the ventral tegmental area (VTA) to the nucleus accumbens (NAc) is represented by a solid dark blue line. The 5-HT2CR is expressed in a greater proportion of parvalbumin-containing GABA interneurons and therefore the 5-HT2CR has its greatest influence (denoted by thick red lines) upon this subtype of GABA neuron compared to GABA neurons that possess calbindin or calretinin, where the 5-HT2CR A exerts minor influence upon activity of these subtypes of GABA neurons (denoted by dotted green and yellow lines, respectively). Stimulation of mPFC 5-HT2CR on parvalbumin-positive GABA interneurons would function to reduce excitatory glutamate output as well as subsequent DA neurotransmission within the mesoaccumbens pathway (VTA and NAc). Although not assessed in this study, the 5-HT2CR may also be expressed in pyramidal neurons (blue hexagon with question mark).

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Serotonin2C receptor localization in GABA neurons of the rat medial prefrontal cortex: implications for understanding the neurobiology of addiction - PubMed (original) (raw)