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Department of Pediatrics, Children's Hospital of Philadelphia and University of Pennsylvania, Philadelphia, Pennsylvania 19104-4318
Submitted 1 August 2003; accepted in final form 19 October 2003
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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; Molliver 1987; Steinbusch 1982). Distinctions in the projections, morphology, neurotransmitter-mediated effects, and electrophysiological characteristics of the 5-HT- and non-5-HT-containing neurons in the MR and DR may be important in understanding the etiology of neuropsychiatric disorders and in understanding the normal regulatory functions of the 5-HT neurotransmitter system.
Both MR and DR send projections to nuclei considered part of the limbic circuit (Azmitia and Segal 1978; Molliver 1987). Distinct projection areas of the DR include prefrontal cortex, lateral septum, and amygdala, whereas MR innervates medial septum, cingulate, and dorsal hippocampus. The DR fibers are small and fine with pleomorphic varicosities, whereas MR fibers are coarse and large with spherical varicosities (Molliver 1987). Neurotoxic 5-HT-releasing agents selectively destroy DR projection fibers without affecting the dense coarse fibers from the MR (Mamounas and Molliver 1988; Mamounas et al. 1991; Molliver et al. 1990). Studies using microdialysis and voltammetry have also indicated that neurotransmitter-mediated responses may be different, and chronic treatment with agonists may differentially regulate the MR and DR (Blier et al. 1990; Kreiss and Lucki 1997; Tao et al. 1996). These studies demonstrate the selective vulnerability of MR or DR, thereby leading credence to the hypothesis that the development and/or treatment of pathological affective states may be due partly to selective alterations at the level of the MR or DR.
The electrophysiological characterization of 5-HT neurons in the DR has been tentative because most studies did not use neurochemical identification. Two studies are primarily cited as a basis for the identification of putative 5-HT-containing neurons. Using in vivo intracellular recording techniques and formaldehyde-induced fluorescence for 5-HT, Aghajanian and Vandermaelen (1982) characterized 5-HT-containing neurons as having a high-input resistance, prominent afterhyperpolarization (AHP) following a single action potential, and long duration action potentials. In a later study that used intracellular recording techniques in a brain slice preparation but did not use neurochemical confirmation, putative 5-HT cells were further defined to have a high-input resistance (150-400 M
), long duration action potential (1.8 ms), and a large, slow AHP (10-20 mV, 200-800 ms). Also cited as a basis for 5-HT neuron identification, but without neurochemical identification, is that the neuron is hyperpolarized by 5-HT1A receptor activation (Aghajanian and Lakoski 1984). Other studies have defined additional characteristics of putative 5-HT neurons (Hajos et al. 1995b, 1996); however, neurochemical identification was not conducted for confirmation.
A primary goal of this study was to combine whole cell recording techniques with immunohistochemical identification of 5-HT-containing neurons to compare the electrophysiological characteristics of DR and MR 5-HT- and non-5-HT-containing neurons. The electrophysiological characteristics of both 5-HT- and non-5-HT-containing neurons in the DR and MR were identified. The majority of the non-5-HT neurons in the DR exhibited characteristics that were very similar to those of 5-HT-containing neurons in the DR. Also, important differences were identified in the characteristics of 5-HT-containing neurons in the MR and DR.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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Electrophysiological recordings
Slices were placed in a recording chamber (Warner Instruments) and continuously perfused with ACSF at 2 ml/min at 32°C maintained by an in-line solution heater (TC-324, Warner Instruments). Neurons were visualized using a Nikon E600 (Optical Apparatus, Ardmore, PA) upright microscope fitted with a 40x water-immersion objective, differential interference contrast (DIC), and infrared filter (IR). The image from the microscope was enhanced using a CCD camera and displayed on a computer monitor. Whole cell recording pipettes were fashioned on a P-97 micropipette puller (Sutter Instruments, Novato, CA) using borosilicate glass capillary tubing (1.2 mm OD, 0.69 mm ID; Warner Instruments). The resistance of the electrodes was 5-10 M when filled with an intracellular solution of (in mM) 130 K-gluconate, 5 NaCl, 1 MgCl2, 0.02 EGTA, 10 HEPES, 2 MgATP, 0.5 Na2GTP, and 0.1% biocytin (pH 7.3).
The DR recordings were confined to the ventromedial DR subdivision that contains the densest cluster of 5-HT neurons. A visualized cell was approached with the electrode, a gigaohm seal was established, and the cell membrane was ruptured to obtain a whole cell recording using an Axoclamp 2A amplifier (Axon Instruments, Foster City, CA). Once the whole cell recording was obtained, cell characteristics were recorded using current-clamp techniques. If the series resistance of the electrode became >20 M, the cell was discarded. Signals were digitized by a Digidata 1320 A/D converter (Axon Instruments) and stored on-line using pClamp 7/8/9 software (Axon Instruments). The liquid junction potential was approximately 10 mV between the pipette solution and the ACSF and was not subtracted from data obtained.
Drugs were added to the ACSF in known concentrations. A stock solution was made and diluted on the day of the experiment to obtain the desired concentration in the ACSF. All chemicals for making the ACSF, electrolyte solution, and 5-carboxyamodotryptamine maleate (5-CT) were purchased from Sigma-Aldrich (St. Louis, MO). WAY 100,635 was generously donated by Wyeth-Ayerst (Princeton, NJ).
Immunohistochemistry
Standard immunofluorescence procedures were used to visualize the filled cell and neurotransmitter content. Slices were fixed by submersion in 4% paraformaldehyde prepared in 0.1 M phosphate buffer (PB; pH 7.4) overnight and stored in 30% sucrose. Sections were incubated with rabbit anti-5-HT antibody (1:2,000, ImmunoStar, Hudson, WI) at 4°C for a period of 48-96 h in the cold room. Initial experiments (approximately one-third of the neurons) used shorter incubation times. We found that with longer incubation times the signal was greater, with no substantial increase in background noise. Subsequently, immunohistochemical labeling was visualized using a FITC (1:100, Jackson ImmunoResearch, West Grove, PA) or Alexa Fluor 488 (1:200, Molecular Probes, Eugene, OR) conjugated donkey anti-rabbit secondary for 60 min at room temperature. The biocytin was visualized using streptavidin conjugated Cy3 (1:1,000 or 1:2,000, Jackson ImmunoResearch) or Alexa Fluor 633 (1:200, Molecular Probes) for 60 min at room temperature. Initial experiments (approximately one-third) used Cy3 and FITC fluorophores. Between incubations, slices were rinsed with PB solutions (3 times for 10 min), and all incubations were done with mild agitation on a shaker. The sections were mounted on superfrost slides and coverslipped with Vectashield mounting medium (Vector Laboratories, Burlingame, CA). Immunofluorescence label was visualized using a Leica DMR fluorescence microscope and a Leica confocal DMIRE2 microscope (Leica). Images were captured using a digital camera and Openlab 3.09 software (Improvision, Lexington, MA) on the fluorescence microscope and a digital camera and Leica Confocal software (Version 2.5, Leica). When using the confocal microscope sequential collection, i.e., Cy3/Alexa 633 separately from FITC/Alexa488, images 0.6 µm in thickness were acquired at the level of the cell body of the biocytin-labeled neuron. The laser power and emission filters were adjusted for both the red and green fluorophor so that there was negligible possibility of false positive result. This was done by exciting at the optimal wavelength for the green fluorophor and detecting using the emission spectra for the red fluorophor and vice versa. When Cy3 and FITC were used, the emission spectra were narrowed on the confocal microscope to reduce the likelihood of bleed-through. Images were adjusted to optimal color balance and contrast using Adobe Photoshop 6.0 software (Adobe, San Jose, CA).
Statistics
ANOVA or t-test was used to test for statistical significance. A probability of P 
0.05 was considered significant. All data are reported as means ± SE.
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RESULTS |
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With the microscope used for electrophysiology, DR neurons were easy to visualize, and they were clustered close together (Fig. 1A). It was not easy to distinguish between 5-HT and non-5-HT cells in the DR based on size or shape. In contrast, in the MR, the cells were very difficult to see; the neurons were not clumped together and there appeared to be dense fiber tracts (Fig. 1B). However, once visualized, the non-5-HT-containing cells were much smaller than the 5-HT-containing cells, and it became relatively easy to differentiate and predict the two cell types based on size in the MR. The differentiation was verified by immunohistochemical identification.
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The non-5-HT-containing neurons could be grouped into three different cell types as seen in Fig. 7. The first type of cell, referred to here as type I (Fig. 7, A-C), had characteristics that were very similar to 5-HT-containing cells. Type I neurons had a large input resistance that was linear (Fig. 6B). The most prominent feature of the second cell type, referred to here as type II (Fig. 7, D-F), was the rectification seen in the voltage responses to hyperpolarizing current pulses and the rebound excitation following the offset of the current pulse (Fig. 7D). The voltage-current plots of type II neurons exhibited a distinct rectification (Fig. 6C). The third cell type, referred to here as type III (Fig. 7, G-I), was differentiated based on the presence of a sag in the voltage responses elicited by hyper-polarizing current pulses. Type III neurons had voltage-current plots that were generated by measuring the peak voltage elicited by the hyperpolarizing current pulse and the voltage at the end of the current pulse just prior to the current pulse termination (Fig. 6D). Whereas the voltage-current plots were linear for the voltage response generated at the beginning of the current pulse, the voltage-current plot for the voltage response at the end of the current pulse rectified. The resistance of the neuron was clearly decreased at the end of the current pulse as measured by the shallow slope.
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In the MR, both 5-HT- and non-5-HT-containing cells were found. As previously stated, it was more difficult to visualize any neuron in the MR compared with the DR. However, when cells were found, it was easier to distinguish the two cell types visually since the non-5-HT cells were smaller in size than the 5-HT cells as seen through the microscope used for electrophysiology. In the MR, 6/14 non-5-HT neurons had characteristics of type I neurons (Fig. 7, A-C), 3/14 non-5-HT-containing neurons exhibited characteristics of type II neurons (Fig. 7, D-F), and 5/14 had characteristics of type III neurons (Fig. 7, G-I). Even though the non-5-HT neurons could be distinguished based on the shape of the voltage-current plots, there were no statistical differences in their cell characteristics, and therefore the data were pooled for comparison to 5-HT-containing neurons in the MR. All of the characteristics were different between the non-5-HT and 5-HT neurons except for the action potential amplitude (Table 1). The membrane resistance of the non-5-HT cells was smaller, the tau was shorter, the AHP amplitude, t1/2, and duration were smaller, and action potential duration was shorter than those of the 5-HT-containing cells. Also, a characteristic that differentiated 5-HT from non-5-HT-containing cell types was the presence of a depolarizing after potential in the AHP of the majority, i.e., 6/14, of the non-5-HT-containing neurons (Fig. 7E). The action potential shape was much different in 5-HT (Fig. 5F) and non-5-HT-containing neurons (Fig. 7, F and I), i.e., much shorter in duration without a shoulder in the falling phase.
The largest category of non-5-HT-containing neurons were type I in both the DR and MR. This category of neuron is characterized by a linear I-V plot. A direct comparison of the characteristics of the type I cells in the DR and MR revealed differences in the input resistance (DR 572 ± 40, MR 335 ± 54; t = 3.15, P = 0.006), tau (DR 41 ± 2.1, MR 19 ± 2.1; t = 7.28, P = 0.000002), AHP t1/2 (DR 126 ± 12, MR 51 ± 18; t = 3.6, P = 0.002), and AHP duration (DR 345 ± 20, MR 194 ± 37; t = 3.84, P = 0.001).
The response to 5-HT1A receptor activation was also measured in both non-5-HT-and 5-HT-containing neurons of the DR and MR. To characterize the 5-HT1A receptor-mediated response, the agonist 5-CT was used. 5-CT has previously been shown to be a potent 5-HT1A receptor agonist (Beck et al. 1992; Williams et al. 1988). In the DR, both the non-5-HT and the 5-HT neurons exhibited a hyperpolarizing response to bath perfusion of 5-CT. The response was significantly smaller in non-5-HT neurons compared with 5-HT neurons (Table 2, Fig. 8). Figure 8 contains two chart recordings, one from a DR non-5-HT-containing neuron (Fig. 8A) and one from a DR 5-HT-containing neuron (Fig. 8B1). These chart recordings show the concentration-dependent hyperpolarization of the membrane potential in response to bath administration of 5-CT. Concomitant with the change in membrane potential was a large decrease in membrane resistance as shown by the decrease in the magnitude of the membrane potential response to the injection of a -30-pA current pulse (downward deflections). There was a significant difference in the magnitude of the response elicited by a 100 nM 5-CT between the non-5-HT- and 5-HT-containing neurons (Table 2). The response in the non-5-HT-containing neurons was around one-half the magnitude of the response elicited in 5-HT-containing neurons. Also, in Fig. 8B2 is a current trace from the same cell in Fig. 8B1, depicting the response to 5-CT (100 nM) administration when the neuron was voltage clamped at -60 mV.
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A direct comparison of the hyperpolarization elicited by different concentrations of 5-CT is summarized in Fig. 10 for the 5-HT-containing neurons of the DR and the MR. The data for the construction of concentration-response curves was collected for 5-HT-containing neurons of the DR and MR using current-clamp techniques. The summarized means of the hyperpolarizing responses elicited by the different concentrations of 5-CT for the DR and MR neurons were fit to a logistic equation to obtain estimates of Emax, EC50, and slope. For the DR neurons, the Emax was equal to 14 mV, EC50 was equal to 7.4 nM, and slope was equal to 1.3. For the neurons in the MR, the Emax was 8.6 mV, the EC50 was 7.0 nM, and the slope was equal to 1.2. The 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. The responses to 100 nM 5-CT were completely blocked by the selective 5-HT1A antagonist WAY 100,635 (0.1 nM) in 5-HT-containing neurons in both the MR (n = 4) and DR (n = 3) and non-5-HT-containing neurons in the DR (n = 4).
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Figure 11 contains current-voltage plots of the outward current elicited by 100 nM 5-CT. The neurons were voltage clamped at -60 mV. A control current-voltage response (Fig. 11, A and C) was elicited by injecting a voltage ramp from -110 to -40 mV (1 mV/s). The 5-CT was administered in the bath, and a current-voltage response was collected again in the presence of 5-CT (Fig. 11, A and C). The amount of current elicited by 5-CT is shown in Fig. 11, B and D, by subtracting the amount of current elicited in the absence of 5-CT from the amount of current elicited in the presence of 5-CT. The reversal potential for neurons recorded from the DR was -90 ± 3.6 mV (n = 6), and from the MR, it was -90 ± 3.7 (n = 4). When the junction potential of 10 mV is taken into account, the reversal potential is approximately -100 mV, in agreement with the estimated Nernst potential, i.e., -103 mV, for potassium, given a [K]o of 2.5 mM and [K]i of 130 mM. The shape of the current-voltage plots and the reversal potentials are similar for the neurons recorded from the MR and DR.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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One of the important findings of this study was the high degree of similarity in the characteristics of the 5-HT and non-5-HT-containing neurons of the DR. Both cell types exhibited the cellular characteristics of a putative 5-HT-containing neuron, in agreement with results we reported previously using intracellular recording techniques (Kirby et al. 2003). Historically 5-HT-containing neurons, as identified by formaldehyde-induced fluorescence techniques, of the DR are characterized by exhibiting a high membrane input resistance, a long time constant, a large afterhyperpolarization, and a broad action potential that has a shoulder on the falling phase (Aghajanian and Vandermaelen 1982). In this study, immuno-histochemistry with whole cell recording was used. All neurons included in this study were identified using immunohis-tochemistry with fluorescent or confocal microscopy. The data from the biocytin-labeled neurons were included only if 5-HT-containing neurons were found in the same plane of focus as the labeled neuron and it was clear that the 5-HT antibody had penetrated to the depth of the biocytin-labeled cell (Figs. 2, 3, 4). In many cases, a neuron that appeared to be unlabeled under the fluorescent microscope was found to be labeled when examined by confocal microscopy. For this reason, all non-5-HT-containing neurons in the DR included in this study were confirmed by confocal microscopy.
The only defining characteristics that differentiated the non-5-HT- and 5-HT-containing neurons in the DR were the tau and the action potential width. Another characteristic that was present in most of the non-5-HT neurons was a depolarizing after potential at the peak of the AHP. The proportion of 5-HT- to non-5-HT-containing neurons is in agreement with estimates in the literature, i.e., approximately one-third to two-thirds (Descarries et al. 1982; Jacobs and Azmitia 1992; Steinbusch et al. 1980, 1981).
One possible explanation for these results is that the non-5-HT cells are 5-HT cells that have stopped producing 5-HT. Previous studies have shown that the levels of the mRNA for the 5-HT synthesizing enzyme tryptophan hydroxylase decreases from postnatal days 22 to 61 (Rind et al. 2000). The decrease is attributed to a loss in 5-HT cells because the density of tryptophan hydroxylase mRNA per cell did not decline with age. It is possible that the levels of the enzyme protein decrease within 5-HT-containing cells; as the rat matures, some cells that made 5-HT at postnatal day 22 no longer make significant quantities of the enzyme and therefore there is a reduction in the amount of neurotransmitter. Even if true, the end result is the same. The non-5-HT-containing neuron that resembles the 5-HT-containing neuron does not produce 5-HT and therefore it will not be secreted as a neurotransmitter in projection areas.
Another explanation that a 5-HT neuron could be misidentified as non-5-HT-containing is that the cell is being dialyzed by the contents of the recording pipette. However, there was no correlation with the length of time of the recording with the 5-HT or non-5-HT nature of the neuron. Many cells that were found to be 5-HT-containing were recorded for prolonged periods of time, i.e., several hours.
Previously there have been reports that the voltage-current or current-voltage plots of the 5-HT-containing neurons of the DR were linear (Crunelli et al. 1983) or demonstrated a degree of rectification (Williams et al. 1988). In this study, the 5-HT-containing cells of both the MR and DR were found to have linear voltagecurrent plots. Also, the majority of the non-5-HT-containing cells of the DR, i.e., type I, were also found to have linear voltagecurrent plots, whereas a minority, type II, demonstrated a nonlinear degree of rectification. The defining characteristic for the 5-HT cells in the study that reported nonlinear rectification is that the neuron exhibit a hyperpolarization in response to 5-CT (Williams et al. 1988). We report here, as well as in a previous study (Kirby et al. 2003), that non-5-HT neurons in the DR also exhibit responses to 5-CT and express 5-HT1A receptors. It is possible that the cells previously identified as 5-HT-containing cells with nonlinear current-voltage plots were in fact non-5-HT-containing neurons.
In contrast, the characteristics of the 5-HT- and non-5-HT-containing neurons in the MR were very different. Even the type I non-5-HT neurons had characteristics that were different from 5-HT-containing neurons in the MR. Also, the type I non-5-HT neurons had different characteristics from the type I non-5-HT neurons in the DR, i.e., membrane resistance, tau, and AHP duration. The classic description of a putative 5-HT neuron compared with a non-5-HT neuron is more appropriately applied to the neurons in the MR than the DR.
The 5-HT1A receptor-mediated hyperpolarization and increase in outward current was also measured in both 5-HT-containing and non-5-HT-containing neurons in the DR and MR. Previously, a 5-HT1A receptor-mediated response was used as one of the defining characteristics of 5-HT-containing neurons (Aghajanian and Lakoski 1984). A 5-HT1A receptor-mediated response was present in the 5-HT-containing neurons in the DR and MR as well as non-5-HT-containing neurons in the DR, but was absent in the non-5-HT-containing neurons in the MR. The magnitude of the response elicited in the non-5-HT-containing neurons in the DR was significantly less than that elicited in the 5-HT-containing neurons. However, these data are important because they confirm our previous report that there are 5-HT1A receptors on non-5-HT-containing neurons in the DR (Kirby et al. 2003) and extend those findings by revealing that non-5-HT-containing neurons in the MR do not have a 5-HT1A receptor-mediated response.
Previous studies have assumed that 5-HT1A receptors were only on 5-HT-containing neurons in the DR. Interpretation and conclusions garnered from these studies may need to be re-evaluated in light of the evidence that some of the neurons may not be 5-HT neurons. Both extracellular and intracellular electrophysiological studies have been conducted looking at the effects of various drug treatments such as antidepressants, corticosterone, behavioral stress paradigms, or 5-HT selective agonists and antagonists on 5-HT1A receptor-mediated responses in putative 5-HT-containing neurons from the DR (Dong et al. 1997; Laaris et al. 1995; Le Poul et al. 1997, 2000; Pineyro and Blier 1999). Putative 5-HT neurons were identified using the criterion defined by the classic studies (Aghajanian and Vandermaelen 1982; Vandermaelen and Aghajanian 1983). The results were discussed in terms of affecting only 5-HT neurotransmission and its role in etiology and treatment of mood disorders. However, the drug effects were most likely also on non-5-HT-containing neurons. The implications are that non-5-HT neurons in the raphe are also affected by drug treatments, and therefore to understand the mechanism of action of the drugs, the effects on both 5-HT- and non-5-HT-containing neurons must be considered.
The concentration-response curve characteristics of 5-CT activation of 5-HT1A receptor were not the same in the DR and MR 5-HT-containing neurons. The efficacy or magnitude of the response was greater in the DR than in the MR, whereas the potency appeared to be the same. These results may be attributed to a difference in the density of the 5-HT1A receptor in the DR and MR or due to a difference in coupling between the 5-HT1A receptor and its second messenger system cascade. Previous studies have reported that the density of 5-HT1A receptors is lower in the MR than DR (Khawaja 1995), but this may be due to the smaller number of cells in the MR as compared with the DR. It is unknown whether the density of the receptors per individual neuron is smaller in the MR compared with the DR. Previous studies, primarily microdialysis studies, have implicated a difference in the efficacy of the 5-HT1A receptor-mediated response between the MR and DR (Casanovas and Artigas 1996; Kreiss and Lucki 1994; Romero and Artigas 1997). However, to our knowledge, no studies have directly measured the 5-HT1A receptor-mediated response in individual 5-HT-containing neurons in the median and dorsal raphe. The importance of these results is that the MR is going to be under less autoreceptor regulation than the DR 5-HT-containing neurons. This difference in the receptor-mediated response also provides a potential site for differential regulation by pathophysiological processes, such as stress, or by drugs, such as antidepressants. These data provide yet another important piece of evidence demonstrating differences between the DR and the MR.
Two of the cellular characteristics were different between 5-HT-containing neurons of the DR and MR. The tau was less in the 5-HT-containing neurons of the MR than the DR. Since the voltage-current plots were linear, the time constant can be used as an indication of the capacitance of the neuron that is a direct consequence of the morphology of the cell (Hille 1992). The difference in the time constant of the MR and DR neurons indicates that there may be differences in their morphology. Also, the processing of synaptic input will be different. Whereas the rise time and amplitude of synaptic input may be greater in the MR, the possibility of synaptic input temporally summating will be less in MR than in the DR neurons. However, it is important to remember that even though whole cell recording techniques were used, the dendritic processes of the 5-HT-containing cells are extensive, and the neuron is not isopotential. The measurement of the neuronal time constant will be primarily influenced by the morphology of the cell near the soma. An in-depth analysis of cell morphology needs to be conducted to test the hypothesis that the morphology of the 5-HT-containing neurons of the MR and DR are different.
The magnitude of the AHP following a single action potential was greater in MR than DR 5-HT-containing neurons. The AHP in the DR is mediated by activation of calcium-dependent potassium channels (Aghajanian 1985; Crunelli et al. 1983; Freedman and Aghajanian 1987). The late phase of the AHP is mediated in part through the release of intracellular calcium through activation of calcium induced calcium release (Pan et al. 1994). The ion channels mediating the AHP in the MR have not been defined. The difference in the magnitude of the AHP between the MR and DR 5-HT-containing neurons could be attributed to many factors. There may be different ion channels that underlie the AHP, a greater density of calcium dependent potassium channels, a difference in channel kinetics, or a larger calcium current. Since there was no difference in the duration of the AHP, the release of intracellular calcium is probably similar in the MR and DR 5-HT-containing neurons. The firing rate of MR neurons is significantly lower than the firing rate of DR neurons (Hajos et al. 1995a). A greater AHP magnitude could lengthen the interspike interval, leading to a lower firing frequency. The greater AHP magnitude in the MR than the DR could be partly responsible for the reported difference in firing frequency.
In conclusion, the important advances of this study are that we examined both 5-HT- and non-5-HT-containing neuron characteristics in the DR and the MR. Three types of non-5-HT-containing neurons were identified. We found that the characteristics of the 5-HT- and non-5-HT-containing neurons of the DR were very similar. In contrast, the differences between the 5-HT and non-5-HT neurons of the MR were numerous. Also, we found important differences in the characteristics of 5-HT-containing neurons in the DR and MR. The tau was smaller and the AHP amplitude larger in 5-HT-containing neurons of the MR compared with those in the DR. Furthermore, a 5-HT1A receptor-mediated response was present in 5-HT-containing neurons of the MR and DR and non-5-HT-containing neurons in the DR, but absent from non-5-HT neurons in the MR. The magnitude of the 5-HT1A receptor-mediated hyperpolarization was larger in 5-HT-containing neurons of the DR compared with the MR. In concert with the evidence that the DR and MR have different projections to forebrain brain regions, these distinctions may be very important in understanding the role of the MR and DR 5-HT circuits in normal physiological processes as well as in the etiology and treatment of pathophysiological states such as affective disorders.
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ACKNOWLEDGMENTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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GRANTS
This work was supported by National Institute of Mental Health Grants MH-60773, MH-63078, and MH-63301.
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FOOTNOTES |
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Address for reprint requests and other correspondence: S. G. Beck, 4 North ARC, Rm. 402A Children's Hospital of Philadelphia, 3615 Civic Center Blvd., Philadelphia, PA 19104-4318 (E-mail: becks{at}email.chop.edu).
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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) 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 voltagecurrent 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 voltagecurrent plot.
