Raphe serotonin neurons are not homogenous: electrophysiological, morphological and neurochemical evidence

Abstract

The median (MR) and dorsal raphe (DR) nuclei contain the majority of the 5-hydroxytryptamine (5-HT, serotonin) neurons that project to limbic forebrain regions, are important in regulating homeostatic functions and are implicated in the etiology and treatment of mood disorders and schizophrenia. The primary synaptic inputs within and to the raphe are glutamatergic and GABAergic. The DR is divided into three subfields, i.e., ventromedial (vmDR), lateral wings (lwDR) and dorsomedial (dmDR). Our previous work shows that cell characteristics of 5-HT neurons and the magnitude of the 5-HT(1A) and 5-HT(1B) receptor-mediated responses in the vmDR and MR are not the same. We extend these observations to examine the electrophysiological properties across all four raphe subfields in both 5-HT and non-5-HT neurons. The neurochemical topography of glutamatergic and GABAergic cell bodies and nerve terminals were identified using immunohistochemistry and the morphology of the 5-HT neurons was measured. Although 5-HT neurons possessed similar physiological properties, important differences existed between subfields. Non-5-HT neurons were indistinguishable from 5-HT neurons. GABA neurons were distributed throughout the raphe, usually in areas devoid of 5-HT neurons. Although GABAergic synaptic innervation was dense throughout the raphe (immunohistochemical analysis of the GABA transporters GAT1 and GAT3), their distributions differed. Glutamate neurons, as defined by vGlut3 anti-bodies, were intermixed and co-localized with 5-HT neurons within all raphe subfields. Finally, the dendritic arbor of the 5-HT neurons was distinct between subfields. Previous studies regard 5-HT neurons as a homogenous population. Our data support a model of the raphe as an area composed of functionally distinct subpopulations of 5-HT and non-5-HT neurons, in part delineated by subfield. Understanding the interaction of the cell properties of the neurons in concert with their morphology, local distribution of GABA and glutamate neurons and their synaptic input, reveals a more complicated and heterogeneous raphe. These results provide an important foundation for understanding how specific subfields modulate behavior and for defining which aspects of the circuitry are altered during the etiology of psychological disorders.

Figure 1. Passive properties of 5-HT and non-5-HT neurons across raphe subfields

(A): Raw trace: Hyperpolarizing and depolarizing current is injected into the cell in a stepwise manner. The cell’s response is measured and its passive characteristics determined. (B) RMP: Across subfields, 5-HT neurons in the dmDR having a more hyperpolarized resting potential compared to vmDR and MR 5-HT neurons (a vs. b: different letters indicate statistical significance between the groups, p<0.05). There was no difference in non-5-HT neurons across subfields. Overall, 5-HT cells have a more hyperpolarized resting membrane potential than non-5-HT cells (solid and dotted lines, p<0.05). Comparing 5-HT and non-5-HT neurons within each specific subfield, 5-HT cells in the dmDR are more hyperpolarized than non-5-HT dmDR cells (ANOVA interaction: F(3,202)=6.05, p<0.001, Newman-Keuls posthoc, *p<0.05). (C) Resistance: Across subfields, resistance of MR 5-HT neurons was significantly greater than that of 5-HT neurons from all other raphe subfields (a vs. b: different letters indicate statistical significance between the groups, p<0.05). Resistance of non-5-HT neurons did not differ across subfields. Overall, 5-HT neurons have a larger resistance compared to non-5-HT neurons (solid and dotted lines, p<0.05). Comparing cell types within a given subfield, MR 5-HT cells have a greater resistance than MR non-5-HT cells (ANOVA interaction, F(3,202)=4.45, p<0.005; Newman-Keuls posthoc, *p<0.05). (D) Tau: 5-HT neurons did not differ across subfields. Among non-5-HT neurons, MR non-5-HT neurons had a lower tau than non-5-HT cells in lw and vmDR (a vs. b: different letters indicate statistical significance between the groups, p<0.05). Tau was usually greater in 5-HT neurons than in non-5-HT cells across subfields (solid and dotted lines, p<0.05). N=number of cells in each group.

Figure 2. Active properties of 5-HT and non-5-HT neurons across raphe subfields

(A): Raw trace: Action potential trace with arrows indicating measurement points used to determine the active properties of the neurons. (B) AP Threshold: Action potential threshold of 5-HT neurons did not differ across subfields. MR non-5-HT neurons possessed a more hyperpolarized action potential threshold than dmDR and vmDR non-5-HT cells (a vs. b: different letter indicates statistical significance between groups, p<0.05). Generally 5-HT neurons had AP thresholds at more depolarized potentials compared to non-5-HT cells (solid and dotted lines, p<0.05). (C) AP Amplitude: Across subfield, AP amplitude of 5-HT lwDR neurons was significantly greater than that of 5-HT neurons in the remaining subfields (a vs. b: different letter indicates statistical significance between groups, p<0.05). AP amplitude was greater in lwDR non-5-HT neurons than non-5-HT cells in any other raphe subfield (c vs. d vs. e: different letter indicates statistical significance between groups, p<0.05). Action potential amplitude was similar between 5-HT and non-5-HT neurons within a particular subfield. (D) AP Duration: For both 5-HT and non-5-HT neurons, AP duration did not differ across subfields. Overall, 5-HT cells have a longer AP duration compared to non-5-HT cells (solid and dotted lines, p<0.05). (E) Activation Gap: Across raphe subfields, the activation gap is larger for 5-HT dmDR cells than 5-HT neurons in any of the other subfields (a vs. b: different letter indicates statistical significance between groups, p<0.05). MR non-5-HT neurons had a smaller activation gap compared to non-5-HT cells in the vmDR (c vs. d: different letter indicates statistical significance between groups, p<0.05). The magnitude of the activation gap is generally greater in 5-HT neurons compared to non-5-HT cells (solid and dotted lines, p<0.001). Comparing within each subfield, the magnitude of the activation gap of dmDR and MR 5-HT cells is greater than that of the non-5-HT cells within the same subfields (ANOVA interaction: F(3,202)=6.19, p<0.01, Newman-Keuls posthoc, *p<0.05). N=number of cells in each group.

Figure 3. Characteristics of the afterhyperpolarization of 5-HT and non-5-HT neurons across raphe subfield

(A) Raw trace: Action potential trace with arrow indicating measurement points used to determine the properties of the AHP. (B) AHP Amplitude: 5-HT in the MR had the largest AHP amplitude (a vs. b), and dmDR 5-HT cells had the smallest (c vs. d) compared to all the other 5-HT neurons (a vs. b, c vs. d, different letters indicate significant differences between groups, p<0.05). AHP amplitude of non-5-HT neurons did not differ across subfields. AHP amplitude was generally greater in 5-HT cells than non-5-HT neurons (solid and dotted lines, p<0.01). Comparing cell types within individual subfields, 5-HT MR cells have a greater AHP amplitude than non-5-HT MR cells (ANOVA interaction, F(3,202)=4.45, p<0.001, Newman-Keuls posthoc, *p<0.05). (C) AHP t(1/2): 5-HT neurons did not differ across subfields. Non-5-HT neurons in the MR had a shorter AHP t(1/2) compared to lwDR and vmDR non-5-HT cells (a vs. b: different letter indicates statistical significance between groups, p<0.05). 5-HT cells have a longer AHP t(1/2) compared to non-5-HT neurons overall (solid and dotted lines, p<0.05). Comparing cell types within the individual subfields, dmDR and MR 5-HT neurons have a greater AHP t(1/2) than non-5-HT cells in the same subfield (*p<0.05). N=number of cells in each group.

Figure 4. 5-CT responses in 5-HT and non-5-HT neurons across subfield

(A) Raw trace: Example trace showing of cell membrane potential. Downward deflections indicate changes in voltage in response to a 300 pA current pulse through the electrode to monitor resistance. The period during which 5-CT was perfused through the recording chamber is indicated by the line above the voltage trace. (B) 5-CT response: Comparing the response to 5-CT in 5-HT neurons across the four subfields, dmDR 5-HT neurons produced a smaller membrane hyperpolarization compared to 5-HT cells in the vmDR (a vs. b: different letter indicates statistical significance between groups, p<0.05). MR and dmDR non-5-HT cells had a smaller response compared to non-5-HT neurons in the lwDR and vmDR (c vs. d: different letters indicate statistical significance between the groups, p<0.05). Comparing 5-HT and non-5-HT neurons, overall vmDR and lwDR show a significantly greater 5-CT response compared to dmDR and MR (*p<0.05). Across all subfields, 5-HT cells produce greater 5-HT_1A response compared to non-5-HT cells (solid and dotted lines, p<0.05). N=number of cells in each group.

Figure 5. GAD67 expression in the DR

GAD67 cells are found ventral to the vmDR in rostral sections and more dorsal locations in the caudal raphe (A1-C1). GAD67 labeling is less intense in the vmDR, lwDR, and dmDR (A2-C2) compared to the surrounding regions (A1-C1; A3-C3). However, although there is little colocalization between GAD67 and 5-HT overall (A1-C1; A2-C2; A3-C3) some colocalization exists between GAD67 and 5-HT, particularly in areas lateral to the vmDR. (A4-C4). Red=GAD67; Green=5-HT.

Figure 6. GAD67 expression in the MR

GAD67 cells were localized to midline and lateral regions of the MR (A1-C1). 5-HT neurons (A2-B2) are interspersed with GAD67 cells across the rostrocaudal extent of MR (A3-C3). Limited co-localization exists between GAD67 and 5-HT (A4-C4). Red=GAD67; Green=5-HT.

Figure 7. GAT1 expression in the DR

In rostral and caudal DR, GAT1 is primarily expressed in areas lateral to vmDR and dmDR (A1, C1). In mid-DR sections, GAT1 is evenly distributed across the DR (B1). Neurons labeled with tryptophan hydroxylase (TPH; A2-B2) are interspersed with GAT1-labeled fibers across the rostrocaudal extent of DR, especially the mid-DR (A3-C3, A4-C4). Confocal images show that GAT1-labeled fibers surround both 5-HT (TPH-labeled, solid arrow) and non5-HT (dashed arrow) neurons (D1-D3). Red=GAT1; Green=TPH

Figure 8. GAT1 expression in the MR

GAT1 was found in regions lateral to the MR (A1-C1). Most TPH-labeled MR neurons (A2-C2) were located along the midline and not within the area of greatest GAT1 expression (A4-C4, D1–D3). Red=GAT1; Green=TPH

Figure 9. GAT3 expression in the DR

GAT3 was expressed evenly throughout the DR (A1-C1). Thus, DR neurons labeled with TPH (A2-C2), across all subfields, were interspersed with GAT3-labeled fibers across the rostrocaudal extent of DR(A3-C3, A4-C4). Confocal images show that GAT3-labeled fibers surround individual TPH-labeled neurons (D1–D3). Red=GAT3; Green=TPH

Figure 10. GAT3 expression in the MR

GAT3 was most strongly expressed in the middle and caudal sections of the MR in both midline and lateral areas (A1-C1). MR neurons labeled with TPH (A2-C2), were interspersed with GAT3-labeled fibers across most of the MR (A3-C3, A4-C4). Confocal images show that GAT3-labeled fibers surround individual TPH-labeled neurons (D1–D3). Red=GAT3; Green=TPH

Figure 11. vGlut3 expression in the DR

In the DR, vGlut3 was observed in all subfields, overlapping with regions TPH is highly expressed (A1-C1, A2-C2, A3-C3). In addition, vgGut3 was expressed in non-5-HT cells lateral to the dmDR and vmDR (A1-C1, A3-C3). TPH cells were interspersed with vGlut3 puncta across the DR (A4-C4). Confocal images show that vGlut3 puncta co-localize with individual TPH-labeled neurons (D1–D3). Red=vGlut3; Green=TPH

Figure 12. vGlut3 expression in the MR

In the MR, vGlut3 was modestly expressed along the midline and had stronger expression in more lateral areas (A1-C1). Some vGlut3 was found interspersed with the population of TPH-labeled neurons (A2-C2, A3-C3). In addition, vGlut3 was expressed in 5-HT (TPH-labeled) and non-5-HT neurons (A4-C4). Red=vGlut3; Green=TPH

Figure 13. Characteristics of the soma of 5-HT neurons across raphe subfields

In all measures (soma volume, surface area, and total area), 5-HT neurons in the vmDR and lwDR had larger cell bodies than 5-HT cells in the dmDR and MR (N=11–14 neurons/group, *p<0.05).

Figure 14. Dendrite length and branching in 5-HT neurons across raphe subfields

(A) The number of primary dendrites (dendrites extending directly from the soma) did not differ across subfield. (B–C) The number of ends and branch points of 5-HT neurons showed a range of variability across the subfields. The dendritic arbor varied between individual neurons within the MR and vmDR, indicating a greater variety in the dendritic architecture of cells within these subfields. In comparison, dendritic arbors of individual neurons within the lwDR and dmDR 5-HT were similar within each subfield. (D) The total dendritic length of neurons in the MR was shorter than that of all the other subfields (*p<0.05). N=11–14 neurons/group.

Figure 15. Polar histogram and Scholl analysis of 5-HT neurons across raphe subfields

(A) Composite polar histograms of neurons from each raphe subfield. Dendritic arbors from neurons in the vmDR and MR tended to extend preferentially in the ventral and dorsal direction. In comparison, lwDR and dmDR neurons did not show a strong directional preference and extended in a wider variety of directions. (A1) Representative traces of neurons from each subfield. Neurons were traced using Neurolucida software and polar histograms generated for each neuron. (A2) A composite tracing of all 5-HT neurons within a particular subfield. Individual tracings were overlayed on top of each other, centered on their cell bodies, and maintained the same scale. (B) Schematic of representative neuron used for Sholl analysis. (B1) Dendrites of MR 5-HT neurons tended to remain closer to the cell body with a sharp decrease in the length of dendrite found in a given radii after 100 μm compared to neurons in the other subfields. (B2) Dendrites of vmDR and MR neurons had a greater number of intersections at radial segments close to the center indicating branching closer to the cell soma. MR had fewer intersections at radial segments further from the center compared to the neurons from the other subfields. Dendrites of lwDR and dmDR neurons had a low number of intersections at radial segments close to the center indicating that dendritic branching occurred further from the soma of these neurons (*p<0.05). N=10–14 neurons/group.

Figure 16. Schematic of rostral, middle, and caudal sections of raphe

Schematic of vGlut3 (pink), GAD67, (blue), GAT1 (red and orange), and GAT3 (yellow and orange) expression overlayed on diagrams of rostral, middle, and caudal raphe. A scale drawing of tracings from vmDR, lwDR, dmDR and MR 5-HT neurons with their dendritic arbor is also included. Note that the drawings are composed of an overlay of all tracings from a specific subfield in order to provide a general idea of the range and direction of dendritic arborization of 5-HT neurons in each subfield. Diagram of raphe sections adapted from Paxinos and Watson (1998).