Glutamatergic drive of the dorsal raphe nucleus

Abstract

The dorsal raphe nucleus (DR) contains the majority of serotonin (5-hydroxytryptamine, 5-HT) neurons in the brain that regulate neural activity in forebrain regions through their widespread projections. DR function is linked to stress and emotional processing, and is implicated in the pathophysiology of affective disorders. Glutamatergic drive of the DR arises from many different brain areas with the capacity to inform the nucleus of sensory, autonomic, endocrine and metabolic state as well as higher order neural function. Imbalance of glutamatergic neurotransmission could contribute to maladaptive 5-HT neurotransmission and represents a potential target for pharmacotherapy. Within the DR, glutamate-containing axon terminals can be identified by their content of one of three types of vesicular glutamate transporter, VGLUT1, 2 or 3. Each of these transporters is heavily expressed in particular brain areas such that their content within axons correlates with the afferent's source. Cortical sources of innervation to the DR including the medial prefrontal cortex heavily express VGLUT1 whereas subcortical sources primarily express VGLUT2. Within the DR, many local neurons responsive to substance P contain VGLUT3, and these provide a third source of excitatory drive to 5-HT cells. Moreover VGLUT3 is present, with or without 5-HT, in output pathways from the DR. 5-HT and non-5-HT neurons receive and integrate glutamatergic neurotransmission through multiple subtypes of glutamate receptors that have different patterns of expression within the DR. Interestingly, excitatory drive provided by glutamatergic neurotransmission is closely opposed by feedback inhibition mediated by 5-HT1A receptors or local GABAergic circuits. Understanding the intricacies of these local networks and their checks and balances, may help identify how potential imbalances could cause psychopathology and illuminate strategies for therapeutic manipulation.

Figure 1. PSD-95 immunolabeling within the mouse DR visualized by array tomography

A. 3D image of the DR rendered from a stack of 28 ultrathin (70nm) serial sections showing immunolabeling for PSD-95 (red), a marker of excitatory synapses, and tryptophan hydroxylase (TPH) (green), to identify serotonin cells. Tissue sections were immunolabeled with rabbit anti-PSD-95, (1:200, Cell Signaling Technologies) and sheep anti-TPH, (1:200, Millipore). B. Arrowheads in A point to same elements in B at higher magnification, showing the exquisitely discrete labeling of the synaptic marker with total absence of out of focus light achieved in array tomography. Scale bars = 50 um in A, and 20 um in B.

Figure 2. Schematic illustration of major glutamatergic afferents to the DR

Black arrows indicate brain regions that provide glutamatergic innervation to the DR including the prefrontal cortex (PFC), lateral habenula (Hb) multiple subregions of the hypothalamus (Hyp), the parabrachial nucleus (PB) and areas in the caudal medulla (CM). As a rule, VGLUT1 (light blue) or VGLUT2 (orange) are predominant in cortical and subcortical domains respectively. There are however exceptions to this rule, depicted as polka-dotted colors (Kaneko et al., 2002; Ziegler et al., 2002).

Figure 3. Cortical afferents to the DR

A subset of the axons that originate in the cortex were identified by the presence of EGFP using piGAP cre-dependent reporter (Badaloni et al., 2007) and Emx1-cre expressing mice (Gorski et al., 2002). EGFP (green) was detected by immunolabeling for GFP (chicken anti-GFP, 1:1000, Aves Labs) and VGLUT1 (red) was detected using guinea pig anti-VGLUT1 (1:1000, Millipore). VGLUT1 is present in the axon varicosities and a few of the double-labeled boutons are indicated with arrowheads. EGFP is only expressed in some cortical neurons (due to mosaic expression of the piGAP reporter) and therefore many VGLUT1 axons (red) lack EGFP. A few VGLUT1-containing EGFP-labeled axons are in proximity to 5-HT cells (arrows). 5-HT cells were identified by immunolabeling for tryptophan hydroxylase 2 (blue; rabbit 1:1000, Novus Biologicals). Scale bar = 12 um.

Figure 4. Schematic view of the most likely location of VGLUT1 and VGLUT2 axons in the DR, with respect to their postsynaptic targets

VGLUT1-containing afferents are more often associated with distal dendrites and spines, whereas VGLUT2 afferents target proximal dendritic shafts and cell bodies. Thus, the two sets of afferent inputs could have different influence on action potential (AP) generation in the postsynaptic cell.

Figure 5. There is a dissociation between colocalization of VGLUT3 and 5-HT in cell bodies vs. axons

Many 5-HT cell bodies have some detectable VGLUT3-immunolabeling (orange), whereas the majority of 5-HT axons in the forebrain lack detectable VGLUT3-immunolabeling (white). Axons containing both VGLUT3 and 5-HT (orange boutons) vary in abundance by brain region. Several areas richly invested with axons containing both VGLUT3 and 5-HT are projection sites of the caudal DR. In addition, there are many VGLUT3 containing cells in the DR that lack 5-HT (pink). These “VGLUT3-glutamate cells” also contribute to ascending projections from the DR (pink). Thus, axons arising from the DR contain 5-HT, VGLUT3, or a combination of both together.

Figure 6. Mapping of VGLUT3-glutamate cells (black dots) in the rat DR from the caudal (A) to rostral (D) pole of the DR

Distances from bregma according to the atlas of Paxinos and Watson (1998) are noted. Figure adapted from (Commons, 2009) reprinted with permission. For each section, a black dot indicates a cell body with immunolabeling for VGLUT3 but not 5-HT; a “VGLUT3-glutamate cell”. Grey dots indicate the location of 5-HT immunolabeled cells (most of which also contain detectable VGLUT3-immunolabeling as depicted in Figure 5). VGLUT3-glutamate cells are located in the center of the nucleus, with a preferential distribution toward the midline as well as mid-dorsoventral locations. Scale bar = 500 um.

Figure 7. Schematic representation of network interactions of VGLUT3-glutamate cells and 5-HT neurons

see text for references. A. Substance P (SP) acting at its receptor, neurokinin 1 (NK1) has the capacity to activate VGLUT3-glutamate neurons (pink). These neurons in turn release glutamate onto some 5-HT neurons (yellow), driving their activation and 5-HT release. This subsequently triggers inhibition of other serotonin neurons via 5-HT1A receptors. This scheme explains experimental observations in the rostral and middle portion of the DR, different relationships may exist in the caudal DR. B. VGLUT3-glutamate neurons also contribute to output projections of the DR, raising the possibility of reciprocal activation states between different output pathways.

Figure 8. Images of glutamate-receptor gene expression in the mouse DR from the Allen Mouse Brain Atlas, Allen Institute for Brain Science, Seattle WA. ©2009. Available from: http://mouse.brain-map.org

Gene expression of ionotropic and metabotropic glutamate receptors in the DR were surveyed and compared to the distribution 5-HT and GABAergic neurons (data summarized in Figure 9). A. Identifying the location of 5-HT cells, TPH2 (tryptophan hydroxylase 2) expression is intense along the midline (arrows). B-E. Glutamate receptors genes that have more expression on the midline than laterally, similar to the pattern of TPH2 expression. F. GAD2 (glutamate decarboxylase-2 or GAD65) expression reveals the distribution of GABAergic neurons in the DR. Cells on the midline (arrow) have lower expression levels than clusters of cells laterally (arrowheads). G-J. Glutamate receptor subunits that, similar to GAD2, show lower expression on the midline (arrows), and higher expression laterally (arrowheads). All panels same scale, bar in A = 400 microns.

Figure 9. Summary of the expression pattern and level of glutamate receptor subunits in the DR as evaluated with the Allen Mouse Brain Atlas, Allen Institute for Brain Science, Seattle WA. ©2009. Available from: http://mouse.brain-map.org (example images depicted in Figure 8)

A dash represents no detectable expression, while circles in increasing sizes proportional to relative abundance when present. The “5-HT Pattern” indicates if the gene was scored as enriched (check mark) or reduced (cross mark) in the regions where 5-HT cells are located. Equivalent expression in 5-HT cell pattern and neighboring areas are unmarked.