Distinguishing characteristics of serotonin and non-serotonin-containing cells in the dorsal raphe nucleus: electrophysiological and immunohistochemical studies

Abstract

The membrane properties and receptor-mediated responses of rat dorsal raphe nucleus neurons were measured using intracellular recording techniques in a slice preparation. After each experiment, the recorded neuron was filled with neurobiotin and immunohistochemically identified as 5-hydroxytryptamine (5-HT)-immunopositive or 5-HT-immunonegative. The cellular characteristics of all recorded neurons conformed to previously determined classic properties of serotonergic dorsal raphe nucleus neurons: slow, rhythmic activity in spontaneously active cells, broad action potential and large afterhyperpolarization potential. Two electrophysiological characteristics were identified that distinguished 5-HT from non-5-HT-containing cells in this study. In 5-HT-immunopositive cells, the initial phase of the afterhyperpolarization potential was gradual (tau=7.3+/-1.9) and in 5-HT-immunonegative cells it was abrupt (tau=1.8+/-0.6). In addition, 5-HT-immunopositive cells had a shorter membrane time constant (tau=21.4+/-4.4) than 5-HT-immunonegative cells (tau=33.5+/-4.2). Interestingly, almost all recorded neurons were hyperpolarized in response to stimulation of the inhibitory 5-HT(1A) receptor. These results suggested that 5-HT(1A) receptors are present on non-5-HT as well as 5-HT neurons. This was confirmed by immunohistochemistry showing that although the majority of 5-HT-immunopositive cells in the dorsal raphe nucleus were double-labeled for 5-HT(1A) receptor-IR, a small but significant population of 5-HT-immunonegative cells expressed the 5-HT(1A) receptor. These results underscore the heterogeneous nature of the dorsal raphe nucleus and highlight two membrane properties that may better distinguish 5-HT from non-5-HT cells than those typically reported in the literature. In addition, these results present electrophysiological and anatomical evidence for the presence of 5-HT(1A) receptors on non-5-HT neurons in the dorsal raphe nucleus.

Fig. 1

Western blot analysis of the 5-HT_1A receptor in rat DRN tissue using the 5-HT_1A receptor antibody. The antibody correctly labels the native, unmodified 5-HT_1A receptor protein with a band at 46 kDa. Two other bands at 35 and 33 kDa likely reflect the breakdown products of the glycosylated form of the receptor, estimated to range from 63 to 70 kDa (see Zhou et al., 1999).

Fig. 2

Immunohistochemistry of a 5-HT-containing (A) and non-5-HT-containing (B) cell in the rat DRN. Fluorescent photomicrograph depicting 5-HT-containing neurons (green) and neurobiotin-filled cell (red). In (A), the cell is double-labeled for both 5-HT and neurobiotin, thus it appears yellow. The neurobiotin-IR cell in (A) is located in the interfascicular subdivision of the DRN (MLF=medial longitudinal fasciculus). The neurobiotin-IR cell in (B) is located in the ventromedial subdivision of the DRN. Scale bar=20 μm in both panels. Images were adjusted for optimal color balance and contrast using Adobe Photoshop, version 6.0.

Fig. 3

Current-voltage responses and time constant (tau) of a 5-HT-containing (A) and a non-5-HT-containing (B) cell in the rat DRN. The lower panels illustrate the membrane potential responses to current steps (580 ms) from −200 pA to 150 pA increasing in increments of 25 pA. The initial portion (indicated by arrows) of the membrane potential response elicited by a −25 pA current pulse (indicated by asterisk) is amplified in the upper panels. A single exponential function is fit to the data in the upper panel and the tau is defined as the time required to reach 63% of the maximum amplitude (ms).

Fig. 4

AHP tau in a 5-HT-containing (A) and a non-5-HT-containing (B) cell in the rat DRN. The AHP tau cell characteristic describes the initial activation phase of the AHP. The upper panels illustrate a single evoked action potential. The outlined portion of the action potential in the upper panels is magnified in the lower panels. A single exponential function is fit to the initial phase of the AHP (from the start of the AHP to its peak, indicated by crosses on lower panels). The AHP tau is defined as the time required to reach 63% of the maximum AHP amplitude (ms).

Fig. 5

Distribution of AHP tau data in 5-HT-containing (N=17) and non-5-HT-containing (N=19) cells in the rat DRN. The AHP tau cell characteristic describes the initial phase of the AHP and is defined as the time required to reach 63% of the maximum AHP amplitude (ms). (A) illustrates the distribution of AHP tau data from individual subjects in the two immunohistochemically-distinguished groups. (B) is a frequency histogram of the AHP tau characteristic in the two immunohistochemically-distinguished groups.

Fig. 6

Similar membrane hyperpolarization response to stimulation of the 5-HT_1A receptor in a 5-HT-IR+ (A) and 5-HT-IR− (B) DRN cell. Chart records indicate the effect of the 5-HT_1A receptor agonist 5-CT (100 nM) on the membrane potential of two immunohistochemically distinct DRN cells. The length of the line above the chart record indicates the length of time that the drug was added to the perfusion medium. Downward deflections in the chart record indicate changes in membrane potential in response to injection of a current pulse (A: 30 pA, 20 s; B: 20 pA, 20 s) through the recording electrode to monitor changes in membrane resistance. The resting membrane potential is −68 mV in the 5-HT-IR+ cell and −66 mV in the 5-HT-IR− cell.

Fig. 7

Fluorescent photomicrographs of 5-HT-containing and 5-HT_1A receptor expressing cells in 30-μm thick coronal sections through the rat DRN. The upper panels (A, D) show 5-HT-immunoreactivity which appears green. The middle panels (B, E) show 5-HT_1A receptor immunoreactivity, which appears red. The bottom panels (C, F) show both 5-HT and 5-HT_1A receptor immunoreactivity such that double-labeled cells appear yellow. The left panels illustrate the distribution of these immunoreactive cells in the ventromedial/interfascicular DRN at low magnification (scale bar=100 μm; MLF=medial longitudinal fasciculus). The right panels are higher magnification photomicrographs of the interfascicular region of the DRN from the left panels (scale bar=20 μm). (F) illustrates cells single-labeled for 5-HT_1A receptor-IR (denoted by asterisks). The remaining cells are double-labeled for 5-HT-IR and 5-HT_1A receptor-IR. Images were adjusted for optimal color balance and contrast using Adobe Photoshop, version 6.0.

Fig. 8

Quantification of 5-HT-containing and 5-HT_1A receptor expressing cells throughout the rostro-caudal extent of the DRN. The number of cells in a particular immunohistochemically-distinct group is represented as a percent of total cells counted in a specific subdivision of the DRN (A: dorsal, B: lateral wings, C: ventral/interfasicular). Cells from one subject were quantified throughout the rostro-caudal extent of the DRN represented by stereotaxic coordinates (in mm) relative to the Bregma coordinate on the rat skull surface (Paxinos and Watson, 1998). 5-HT-IR cells are represented by black bars, 5-HT_1A receptor-IR cells are represented by white bars, and cells double-labeled for both 5-HT-IR and 5-HT_1A receptor-IR are represented by gray bars.

Fig. 9

Distribution of 5-HT_1A receptor-IR labeling in the septum (A1 and A2), supraoptic nucleus (B1 and B2) and medial habenula (C). (A) illustrates the distribution of 5-HT_1A receptor-IR labeling in the septum at low (A1; scale bar=300 μm; LV=lateral ventricle, LS=lateral septum, MS=medial septum) and high magnification (A2; scale bar=50 μm). (B) illustrates the distribution of 5-HT_1A receptor-IR labeling in the supraoptic nucleus at low (B1; scale bar=100 μm; ox=optic chiasm) and high magnification (B2; scale bar=50 μm). (C) illustrates the distribution of 5-HT_1A receptor-IR labeling in the medial habenula (scale bar=50 μm; 3V=third ventricle). Arrows in panels A1, A2, B1, and B2 indicate labeled cells in corresponding high and low magnification photomicrographs. Images were adjusted for optimal brightness and contrast using Adobe Photoshop, version 6.0.