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Comparative Study
, 91 (2), 994-1005

Median and Dorsal Raphe Neurons Are Not Electrophysiologically Identical

Affiliations
Comparative Study

Median and Dorsal Raphe Neurons Are Not Electrophysiologically Identical

Sheryl G Beck et al. J Neurophysiol.

Abstract

The dorsal (DR) and median raphe (MR) nuclei contain 5-hydroxytryptamine (serotonin, 5-HT) cell bodies that give rise to the majority of the ascending 5-HT projections to the forebrain limbic areas that control emotional behavior. In the past, the electrophysiological identification of neurochemically identified 5-HT neurons has been limited. Recent technical developments have made it possible to re-examine the electrophysiological characteristics of identified 5-HT- and non-5-HT-containing neurons. Visualized whole cell electrophysiological techniques in combination with fluorescence immunohistochemistry for 5-HT were used. In the DR, both 5-HT- and non-5-HT-containing neurons exhibited similar characteristics that have historically been attributed to putative 5-HT neurons. In contrast, in the MR, the 5-HT-and non-5-HT-containing neurons had very different characteristics. Interestingly, the MR 5-HT-containing neurons had a shorter time constant and larger afterhyperpolarization (AHP) amplitude than DR 5-HT-containing neurons. The 5-HT(1A) receptor-mediated response was also measured. The efficacy of the response elicited by 5-HT(1A) receptor activation was greater in 5-HT-containing neurons in the DR than the MR, whereas the potency was similar, implicating greater autoinhibition in the DR. Non-5-HT-containing neurons in the DR were responsive to 5-HT(1A) receptor activation, whereas the non-5-HT-containing neurons in the MR were not. These differences in the cellular characteristics and 5-HT(1A) receptor-mediated responses between the MR and DR neurons may be extremely important in understanding the role of these two 5-HT circuits in normal physiological processes and in the etiology and treatment of pathophysiological states.

Figures

FIG. 1
FIG. 1
Photomicrograph taken through the microscope used for electrophysiology of the dorsal raphe (A) and median raphe (B). In both panels, a recording electrode can be seen attached to a neuron. In B, the neuron is indicated by an arrow. Scale bar, 50 μm.
FIG. 2
FIG. 2
Photomicrographs of a 5-hydroxytryptamine (5-HT)-Containing dorsal raphe (DR) neuron. Fluorescent photomicrographs taken on the confocal microscope depicting green 5-HT–containing neurons (A1–C1), a red biocytin-filled cell (A2–C2), and the overlay of the 2 (A3–C3). A z stack of 79 photomicrographs 0.6 μm in thickness were taken, extending through 51 μm of tissue. In A1–A3, the photomicrograph is in the x-y axis; in B1–B3, the photomicrographs are in the y-z axis; and in C1–C3, the photomicrographs are in the x-z axis. Scale bar in A2, 20 μm. Arrow points to the biocytin-filled cell that is also 5-HT immunoreactive.
FIG. 3
FIG. 3
Photomicrographs of a non–5-HT–containing DR neuron. Fluorescent photomicrographs taken on the confocal microscope depicting green 5-HT–containing neurons (A1–C1), a red biocytin-filled cell (A2–C2), and the overlay of the 2 (A3–C3). A z stack of 37 photomicrographs 0.6 μm in thickness were taken, extending through 22 μm of tissue. In A1–A3, the photomicrograph is in the x–y axis; in B1–B3, the photomicrographs are in the y–z axis; and in C1–C3, the photomicrographs are in the x–z axis. Arrowhead points to the non–5-HT–containing biocytin-filled cell and the black space where the cell should appear if it was 5-HT immunoreactive. Arrow points to a different neuron in the same plane of focus as the biocytinfilled cell that contains 5-HT. Note that the 5-HT immunoreactivity appears throughout the extent of the z axis in B1 and C1. Scale bar in A2, 20 μm.
FIG. 4
FIG. 4
Immunohistochemistry of a 5-HT–containing and non–5-HT–containing cell in the medial raphe (MR). In A1–C3, the photomicrographs were taken at the plane of focus for the neuron that is on the right of A1–A3, whereas D1–F3 were taken at a plane of focus for the neuron on the left of D1–D3. A z stack of 119 photomicrographs 0.6 μm in thickness were taken, extending through 72 μm of tissue. The x-y axis is shown in A1–A3 and D1–D3, the y-z axis in B1–B3 and E1–E3 and the x-z axis in C1–C3 and F1–F3. Fluorescent photomicrograph depicting red biocytin-filled cells (A2, B2, C2, D2, E2, and F2), green 5-HT–containing neurons (A1, B1, C1, D1, E1, and F1), and the overlay of the two (A3, B3, C3, D3, E3, and F3). The arrow indicates the cell body and process of the neuron (A1, B1, and C1) that appears orange and is therefore double-labeled for biocytin and 5-HT in A3, B3, and C3. Arrowhead in D2, D3, E1, F1, and F2 indicates the cell body and process of the neuron that is not double-labeled. The arrow in D1, D3, and F1 demarcates a 5-HT–containing neuron in the same x plane of focus of the biocytin-labeled neuron.
FIG. 5
FIG. 5
Characteristics of 5-HT–containing neurons of the DR (A–C) and MR (D–F). In A and D, hyperpolarizing and depolarizing current pulses were injected into the cell, and the voltage response was measured. In B and E, a depolarizing current pulse of sufficient magnitude to elicit a single action potential was used to generate an afterhyperpolarization following the action potential. In C and F, a current pulse of sufficient magnitude was used to generate a single action potential. In A, B, D, and E, the action potentials are truncated due to the sampling rate.
FIG. 6
FIG. 6
Voltage–current plots for a 5-HT–containing neuron from the DR (A) and non–5-HT–containing neurons of type I from the DR (B), type II from the MR (C), and type III from the MR (D). Data were obtained by measuring the maximum voltage response to hyperpolarizing current pulses. In D, maximum voltage response (●) and the voltage at the end of the current pulse (■) were measured, due to the sag in the membrane potential during the hyperpolarizing current pulse. In each case, the voltage–current plot was fit by a straight line to obtain measurements of input resistance and resting membrane potential. In C, only the 1st 4 points were fit due to rectification of the voltage–current plot.
FIG. 7
FIG. 7
Three different non–5-HT neuron types. In A–C, characteristics of a type I neuron from the DR is shown; in D–F, characteristics of a type II neuron from the MR is shown; and in G–I, characteristics of a type III neuron from the MR are shown. In A, D, and G, voltage responses to injection of hyperpolarizing and depolarizing current pulses are plotted. In B, E, and H, afterhyperpolarizations following a single action potential are shown; and in C, F, and I, action potentials are shown. Note the presence of a depolarizing afterpotential at the peak of afterhyperpolarization in B and E. In A, B, D, E, G, and H, action potentials are truncated due to sampling rate.
FIG. 8
FIG. 8
Membrane potential responses to bath perfusion of 5-carboxyamodotryptamine maleate (5-CT) in non–5-HT–containing (A) and 5-HT–containing (B1) neurons in the DR. B2: chart recording of current elicited by bath application of 5-CT as recorded in voltage clamp with the cell held at −60 mV. Length of line above each chart recording depicts the amount of time the drug was in the chamber. Downward deflections are voltage response to intracellular injection of a −30-pA current pulse used to monitor changes in membrane resistance. Resting membrane potential for cell A was −72 mV and for cell B was −56 mV.
FIG. 9
FIG. 9
Membrane potential responses to bath perfusion of 5-CT in non–5-HT–containing (A) and 5-HT–containing (B) neurons in the MR. Downward deflections are voltage response to intracellular injection of a −30-pA current pulse used to monitor changes in membrane resistance. Length of line above each chart recording depicts the amount of time the drug was in the chamber. Resting membrane potential for cell A was −58 mV and for cell B was −63 mV.
FIG. 10
FIG. 10
Concentration–response curves for the averaged data of the neurons recorded in the DR (squares) and the MR (triangles). Number of observations per data point is presented in parentheses on graph. Data were fit to a logistic equation to obtain estimates for EC50, Emax, and slope. For the DR neurons, the Emax was equal to 14 mV, EC50 was equal to 7.4 × 10−9 M, and slope was equal to 1.3. For the neurons in the MR, the Emax was 8.6 mV, the EC was 7.0 × 10−9 50 M, and slope was equal to 1.2. Response elicited by 100 nM 5-CT (t = 2.31; P = 0.03; n = 16 for DR and 10 for MR) was significantly less in 5-HT–containing cells of the MR compared with the neurons of the DR.
FIG. 11
FIG. 11
current–voltage plots for 5-CT (100 nM) activation of 5-HT1A receptor-mediated outward current in 5-HT–containing neurons of the DR (A and B) and MR (C and D). In A and C, current–voltage plots elicited by voltage ramp from −110 to −40 mV (1 mV/s) in the absence and presence of 5-CT. In B and D, current elicited by 5-CT obtained by subtracting amount of current in the absence of 5-CT from the current elicited in the presence of 5-CT.

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