Serotonin Induces EPSCs Preferentially in Layer V Pyramidal Neurons of the Frontal Cortex in the Rat

E.K. Lambe, P.S. Goldman-Rakic and G.K. Aghajanian

Interdepartmental Neuroscience Program, Section of Neurobiology, and Departments of Psychiatry and Pharmacology, Yale University School of Medicine, New Haven, CT, USA


    Abstract
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Notes
 References
 
The effect of serotonin (5-HT) on the release of glutamate was examined in pyramidal cells in layers II–VI of the frontal cortex. The intracellular recording electrode contained 1% biocytin so the neurons could later be visualized with an avidin-biotin peroxidase method. Pyramidal cells in layer V of the frontal cortex showed the greatest 5-HT-induced increase in both the frequency and amplitude of ‘spontaneous’ (non-electrically evoked) excitatory post-synaptic currents (EPSCs). A small proportion of neurons in layer II/III showed an increase in EPSC frequency, whereas cells in layer VI showed no significant change in either EPSC frequency or amplitude. The physiological response to 5-HT mirrors the high density of 5-HT2A receptors in layer V, as well as the pattern of thalamic projections in frontal cortex. The specific induction of EPSCs in layer V neurons suggests that 5-HT preferentially modulates the output neurons of the frontal cortex.


    Introduction
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Notes
 References
 
Previous studies suggest that serotonin (5-HT)2A receptors modulate excitatory transmission in cortical circuits which may account for normal fluctuations in mood, attentional state and capacity for information processing (Marek and Aghajanian, 1998bGo). Activation of the 5-HT2A receptor is thought to be responsible for the psychotomimetic action of psychedelic hallucinogens (Glennon et al., 1984Go; Rasmussen and Aghajanian, 1986Go; Egan et al., 1998Go; Vollenweider et al., 1998Go), and blockade of this receptor has been shown to be a critical component in the action of atypical antipsychotic drugs (Breier, 1995Go; Lieberman et al., 1998Go; Meltzer and Nash, 1991Go).

A striking effect of 5-HT is to increase the frequency and amplitude of ‘spontaneous’ (non-electrically evoked) excitatory post-synaptic currents (EPSCs) in neurons recorded in vitro in the mid-layers of the frontal cortex. The modulation of glutamate release is mediated by the activation of 5-HT2A receptors in the cerebral cortex and can be blocked by MDL 100907, an antagonist selective for the 5-HT2A receptor (Aghajanian and Marek, 1997Go). While the prefrontal neurons displaying 5-HT-evoked EPSCs were presumed to be layer V pyramidal cells (Aghajanian and Marek, 1997Go), their morphology and laminar position have not been identified and confirmed with anatomical methods. The increase in EPSC amplitude may be due to a postsynaptic mechanism, whereas a presynaptic mechanism appears to underlie the 5-HT-induced increase in EPSC frequency. Although the 5-HT-induced EPSCs can be blocked by adding tetrodotoxin or by removing calcium from the bath, they do not appear to be impulse driven since neurons in the brain slice are rarely induced to fire by bath application of 5-HT (Marek and Aghajanian, 1998bGo). Instead, the 5-HT-induced increase in EPSCs appears to result from a focal mechanism involving an increase in glutamate release from excitatory nerve terminals impinging upon the apical dendrite (Aghajanian and Marek, 1997Go).

Thalamic afferents have been suggested as the possible source of these excitatory axon terminals which release glutamate onto the apical dendrites of layer V neurons (Marek and Aghajanian, 1998aGo). The µ-opiate receptor agonist DAMGO completely suppresses 5-HT2A-induced EPSCs in the medial prefrontal cortex. Since cortical glutamatergic cells do not appear to express µ-receptor mRNA (Mansour et al., 1994Go), this suppression suggests that 5-HT induces glutamate release from subcortical afferents in the frontal cortex (Marek and Aghajanian, 1998aGo). Among the glutamatergic efferents to the cerebral cortex, the neurons in the thalamus are notable in that they contain substantial levels of mRNA for the µ-opioid receptor (Mansour et al., 1994Go). Moreover, there appears to be a coincidence in the laminar distribution of 5-HT2 receptors and a specific subset of thalamocortical projections in the cortex. An autoradiographic binding study by Blue et al. showed that layer I and superficial layer V have dense bands of 5-HT2 receptors (Blue et al., 1988Go), and more recent studies have shown a high density of 5-HT2A receptor immunoreactivity on the apical dendrites of layer V neurons and, to a lesser extent, on nearby presynaptic terminals (Willins et al., 1997Go; Hamada et al., 1998Go; Jakab and Goldman-Rakic, 1998Go). Similarly, the midline and intralaminar nuclei of the thalamus project mainly to layers I and superficial V of the frontal cortex (Berendse and Groenwegen, 1991).

To determine whether 5-HT-induced EPSCs in the neocortex are specific to layer V neurons, we measured the effect of a near-maximal concentration of 5-HT on EPSC frequency and amplitude in different layers of the rat frontal cortex using biocytin-filled electrodes to allow the recorded cells to be visualized and their laminar position to be determined by comparison with adjacent Nissl-stained sections.


    Materials and Methods
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Notes
 References
 
In brief, intracellular recording in pyramidal cells of the rat frontal cortex was used to examine the effect of 5-HT on the release of glutamate across the cortical width, as indicated by changes in the frequency of spontaneous EPSCs.

Preparation of cortical slice

Male rats (50–170 g) were deeply anesthetized with choral hydrate (400 mg/kg) and then decapitated. The brains were removed, placed in ice-cold, oxygenated artificial cerebrospinal fluid (ACSF) with equimolar sucrose substituted for NaCl (sucrose-ACSF) (Aghajanian and Rasmussen, 1989Go) and blocked perpendicular to the cortical surface to ensure that apical dendrites from cells in layer V remained intact. Coronal slices (500 µm thick) of the anterior frontal cortex were cut in sucrose-ACSF using a microslicer (DSK, Dosaka, Japan) and placed in an interface-type chamber. The standard ACSF (pH 7.35) used for slice perfusion was oxygenated with 95% O­2/5% CO2 and contained the following: 128 mM NaCl, 3 mM KCl, 1.25 mM NaH2PO4, 10 mM D-glucose, 25 mM NaHCO3, 2 mM CaCl2 and 2 mM MgSO4. The slice was heated slowly from room temperature to ~33°C. There was a recovery period of 2 h prior to beginning the experiment.

Intracellular Recording

Sharp electrodes containing 1 M potassium acetate (32–80 M{Omega}) were used to record from cells in current or voltage clamp. This non-chloride-containing electrode solution was selected to ensure conditions preferential for the detection of EPSCs and contained 1% biocytin for the later visualization of the recorded neurons with avidin-biotin complex binding (ABC; Standard Elite kit, Vector, Burlingame, CA) followed by a diaminobenzidine (DAB) reaction. The holding potential was near the ECl and Vrest for the cells: –70 to –80 mV. Under these conditions, post-synaptic currents have been shown to be completely blocked by AMPA/kainate antagonists (Aghajanian and Marek, 1997Go), indicating that they represent glutamatergic EPSCs. The baseline and 5-HT-induced EPSCs were recorded in the discontinuous single-electrode voltage clamp mode of the Axoclamp 2-A (Axon Instruments, Foster City, CA) at a switching frequency of ~6 kHz. Cells were recorded in medial prefrontal [Cg1, Cg2, Cg3 (Zilles, 1985Go)] and frontoparietal [Fr1, Fr3, Par1 (Zilles, 1985Go)] cortex.

Application of 5-HT

5-HT was bath-applied at 100 µM in ACSF. Previous work (Aghajanian and Marek, 1997Go) has shown that this concentration gives a near-maximal increase in EPSC frequency. Exposure to 5-HT was limited to 1 min and followed by at least a 5 min wash-out in order to avoid desensitization.

Data Collection and Analysis

P-Clamp software (Axon Instruments) via a Digidata 1200 interface was used for data collection. Cell and spike characteristics were measured using Clampfit software (Axon Instruments). EPSC frequency and amplitude were measured with the Mini Analysis Program software (Synaptosoft, Leonia, NJ). Statistical comparisons of changes in each cell's response to 5-HT with made using the non-parametric Kolmogorov-Smirnov two-tailed test for distributions (Goodman, 1954Go), with a significance criterion of P = 0.01. A one-factor analysis of variance (ANOVA) followed by post-hoc Scheffé F-tests were used to make statistical comparisons of the frequency changes from baseline for the three layers.

Histochemistry

As described above, the electrodes were filled with a 1 M potassium acetate solution containing 1% biocytin. After the cell characteristics and response to 5-HT were measured, the cells were held for an additional 15–30 min to allow for diffusion of biocytin to the distal portions of the apical and basal dendrites. To allow visual confirmation of the laminar position of each cell, electrode penetrations were minimized and a maximum of three cells per slice were tested and filled. To establish that the slice was in good condition (based on preliminary evidence that hypoxia decreases the 5-HT response), each layer II/III or layer VI cell was obtained only after a layer V cell had been confirmed to have an increase in EPSC frequency after bath application of 100 µM 5-HT.

The agranular nature of the medial prefrontal cortex, which lacks a discernible layer IV, makes the distinction between layers II/III and V difficult to resolve. This distinction is much clearer in the lateral regions of frontal cortex. Since there is a sizeable response to 5-HT in the lateral as well as medial cortex (Aghajanian and Marek, 1997Go), cells were selected in both of these areas. The boundary between layers II and III is indistinguishable in both the medial and lateral frontal cortex of the rat.

Each slice was immediately post-fixed in 4% paraformaldehyde for 48 h at 4°C. Slices were cryoprotected in 20% sucrose solution and then resectioned to 80 µm on a freezing microtome. The sections were incubated for 3 h in serum solution with Triton X-100 to allow permeation and to block nonspecific binding. After several washes, the sections were incubated with ABC (Standard Elite kit) at 4°C overnight. A nickel-intensified DAB reaction was used to visualize the biocytin-filled cells. Sections were dehydrated in alcohol and cleared in xylene before being mounted on glass slides with cytoseal. Nissl staining of an adjacent section was used to confirm the laminar distribution of the filled cells.


    Results
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Notes
 References
 
The characteristics of the 24 recovered cells are described in Table 1Go. There were no significant differences across the layers in electrophysiological properties, including resting potential, input resistance and spike properties, recorded in current clamp mode. Baseline EPSC frequency and amplitude were recorded in voltage clamp mode, with the holding potential near the resting Vm. The effect of 1 min of application of 100 µM 5-HT was then determined, as shown in Table 2Go. The intracellular marking and histochemical visualization were successful in all layers, as pictured in Figure 1ACGo. The layer of the cells was verified with Nissl staining in adjacent sections, as shown in Figure 1A.3 and C.3Go. Neurons from different layers could be easily distinguished on the basis of morphology and by comparison with an adjacent Nissl-stained section. As illustrated in Figure 1Go, layer II/III neurons had relatively short apical dendrites which extended to layer I, whereas the layer V neurons had apical dendrites which could be followed (sometimes through many sections) to layer I.


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Table 1 Electrophysiological characteristics
 

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Table 2 Excitatory post-synaptic currents
 


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Figure 1.  The intracellular marking and histochemical visualization were successful in all layers. Photomicrographs A.1, B.1 and C.1 show typical examples at x20 magnification of neurons in layer II/III, layer VI and layer V respectively (scale bar = 50 µm). The laminar position of these cells is shown in A.2–C.2 (scale bar = 200 µm). The latter images can be compared with Nissl-stained sections A.3 (lateral) and C.3 (medial).

 
Recordings from the cells in Figure 1Go are illustrated in Figure 2Go, first at baseline and then during the bath application of 5-HT. Of the recovered cells in layer V, 7/8 (87%) had significant increases in EPSC frequency and 6/8 (75%) showed significant increases in EPSC amplitude, as assessed by the Kolmogorov-Smirnov test of distributions. In layer II/III, only 2/8 (25%) cells showed significant increases in EPSC frequency, although several others showed nonsignificant trend increases. None of the layer II/III cells showed significant increases in EPSC amplitude. None of the layer VI neurons displayed increases in EPSC frequency. No layer VI neuron showed a significant increase in EPSC amplitude and 1/8 (13%) neurons showed a significance decrease. The Kolmogorov-Smirnov tests of inter-event interval and amplitude from the cells in Figures 1Go and the voltage sweeps in Figure 2Go are shown in Figure 3Go.



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Figure 2.  The typical effects of serotonin on the basal frequency and amplitude of EPSCs are illustrated above. Intracellular recording in voltage clamp show six consecutive 1 s episodes under basal conditions and during the application of 100 µM 5-HT. There is a robust increase in the frequency and amplitude of spontaneous EPSCs in the layer V neurons, but not in the layer II/III or layer VI neurons.

 


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Figure 3.  Normalized cumulative distributions before and during the application of 5-HT (data taken from ten 1 s episodes for each of the cells illustrated above). The shifts in distribution to higher frequency and higher amplitude were significant (P = 0.0001) only for the layer V cell (Kolmogorov-Smirnov test).

 
The laminar differences in the response to the bath application of a near-maximal concentration of 5-HT are summarized in Figure 4Go. A one-factor ANOVA was used to compare the frequency change from baseline for the three layers [F = 9.43; P = 0.001]. Post-hoc Scheffé F-tests showed that the 5-HT- induced increase in EPSC frequency seen in layer V neurons was significantly higher than that seen in either layer II/II or layer VI.



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Figure 4.  Laminar difference in the response to bath application of 5-HT. The change in mean frequency (± SEM) in response to 5-HT (100 µM, 1 min) for the entire sample of cells tested in each layer (n = 8 per layer). A one-factor ANOVA was used to compare the frequency change from baseline for the three layers *F = 9.43; P = 0.001.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Notes
 References
 
The key finding of this study is that layer V pyramidal neurons differ from pyramidal cells in other layers of the frontal cortex. These neurons consistently show significant increases in both EPSC frequency and amplitude upon bath application of 5-HT. By contrast, only a small fraction of layer II/III cells, and no layer VI neurons, show a significant increase in EPSC frequency in response to a maximal concentration of 5-HT.

Receptor Localization

These findings are consistent with an earlier autoradiography study showing a dense band of 5-HT2A receptor binding in superficial layer V of both medial and lateral frontal cortex (Blue et al., 1988Go). Recent immunohistochemical studies in rat have demonstrated a particularly high density of 5-HT2A receptors in layer V cells, along their apical dendrites, and in the adjacent neuropil, although there is uncertainty about the degree of membrane insertion and the presence of 5-HT2A receptors in spines (Willins et al., 1997Go; Hamada et al., 1998Go; Cornea-Hébert et al., 1999Go). The localization is consistent with an electrophysiological study showing ‘hot spots’in the vicinity of the apical dendrite — locations where the iontophoresis of 5-HT increases EPSC frequency at the soma (Aghajanian and Marek, 1997Go). Immunohistochemical and autoradiographic analyses of 5-HT2A receptors in monkey and human neocortex also show an intense band of labeling in layer V, but with additional label in layer III (Burnet et al., 1995Go; Pasqualetti et al., 1996Go; Jakab and Goldman-Rakic, 1998Go). This finding raises the question of how 5-HT would affect spontaneous EPSCs in layer III pyramidal cells of primate neocortex. However, a recent study by Newberry et al. (Newberry et al., 1999Go) found that 5-HT did not increase the frequency or amplitude of EPSCs in layer III of human frontal cortex (Newberry et al., 1999Go), suggesting that to this extent there is similarity in the laminar distribution of the 5-HT response across species. While most of the 5-HT2A receptor is located postsynaptically, there is also evidence for a presynaptic location (Jakab and Goldman-Rakic, 1998Go; Leysen et al., 1982Go). Differences between the immunohistochemical and physiological localization would be expected if increase in EPSCs results from the activation of a lesser population of presynaptic 5-HT2A receptors.

Projections to the Frontal Cortex

Previous work suggests that the activation of the 5-HT2A receptor modulates glutamate release from only a subset of glutamatergic projections to the apical dendrites of layer V pyramidal cells (Marek and Aghajanian, 1998aGo). The 5-HT-induced EPSCs appear to result from increased glutamate release from local terminals rather than an increase in impulse flow (Aghajanian and Marek, 1997Go), since the relevant cell bodies are not in the slice. This conclusion is based partly on the fact that agonists of µ-opiate receptors can completely suppress the 5-HT-induced increases in EPSC frequency. Since cortical neurons do not appear to express µ-receptor mRNA (Mansour et al., 1994Go), and since removal of all afferents to anterior cingulate cortex results in a dramatic decrease in µ-receptor binding (Vogt et al., 1995Go), the relevant glutamatergic terminals are likely subcortical in origin (Marek and Aghajanian, 1998aGo). The most likely sources are the midline and intralaminar nuclei which project to layers I and superficial layer V of the frontal cortex (Berendse and Groenwegen, 1991), layers which have dense bands of 5-HT2 receptor. While mRNA expression has been found for the 5-HT2A receptor in the midline thalamus (Pazos and Palacios, 1985Go; Appel et al., 1990Go) and 5-HT2A receptor protein has been found in transport along axonal tracts (Cornea-Hébert et al., 1999Go), co-localization of 5-HT2A and µ-opioid receptors on thalamocortical axon terminals has not yet been examined. Ultrastructural analysis has revealed that the intralaminar thalamic projections form asymmetrical synapses on the spines of pyramidal cell dendrites (Marini et al., 1996Go).

Larkman has shown that most pyramidal cells have the highest density of spines distal to the initial segment of the apical dendrite (Larkman, 1991Go). In layer V pyramidal cells, this region of highest spine density tends to fall in superficial layer V, the region of maximal 5-HT2A receptor density and maximal density of projections from the midline and intralaminar nuclei of the thalamus. This is the same region in which EPSC frequency is highly sensitive to the iontophoresis of 5-HT (Aghajanian and Marek, 1997Go).

Layer V Projections

The laminar specificity of the 5-HT-induced increase in EPSC frequency is particularly interesting in view of the difference in projections from the different layers. Whereas layer II/III projections tend to be directed to other areas of cortex and layer VI projections mainly target the thalamus as well as some areas of the cortex (DeFelipe and Fariñas, 1992Go; Groenwegen et al., 1997), layer V pyramidal cells are output neurons with diverse projections to subcortical structures. These neurons have been shown to project to the basal ganglia (Gerfen, 1989Go; Levesque et al., 1996Go; Groenewegen et al., 1997Go) and the thalamus (Giguere and Goldman-Rakic, 1988Go; Schwartz et al., 1991Go; Deschenes et al., 1994Go), as well as many nuclei in the brainstem and midbrain (Neafsey et al., 1986Go; Ferino et al., 1987Go; Terreberry and Neafsey, 1987Go; Zaborszky et al., 1997Go; Hajós et al., 1998Go; Peyron et al., 1998Go) and spinal cord (Deschenes et al., 1994Go; Levesque et al., 1996Go). Layer V neurons in the medial frontal cortex appear to be involved in determining overall cortical tone and activation levels with projections to the periaquaductal gray matter (Neafsey et al., 1986Go), as well as the monoamine-producing nuclei (Dalsass et al., 1981Go; Arnsten and Goldman-Rakic, 1984Go; Luppi et al., 1995Go; Hajós et al., 1998Go; Peyron et al., 1998Go; Juckel et al., 1999Go). The medial frontal cortex is the only source of cortical efferents to the raphe (Aghajanian and Wang, 1977Go; Hajos et al., 1998Go; Peyron et al., 1998Go) and locus coeruleus (Cedarbaum and Aghajanian, 1978Go; Luppi et al., 1995Go), which respectively produce 5-HT and norepinephrine. Layer V neurons in the medial frontal cortex have also been demonstrated to play a major role in the affective modulation of the visceral or autonomic nervous system (Terreberry and Neafsey, 1987Go).

Function

The induction of EPSCs by 5-HT in pyramidal cells of the frontal cortex has previously been shown to be mediated by 5-HT2A receptors (Aghajanian and Marek, 1997Go; Marek and Aghajanian, 1999Go). Psychedelic hallucinogens are potent agonists at the 5-HT2A receptor in the cortex and produce a psychotic syndrome in healthy controls, which in some respects is comparable to that seen in acutely ill patients with schizophrenia (for further discussion of the controversial issues in this comparison, see Gouzoulis-Mayfrank et al. (Gouzoulis-Mayfrank et al., 1998Go)]. The effects of hallucinogens, such as psilocybin, include disturbances of spatial and temporal perception, deficits in working and short-term memory, and cognitive dysfunction affecting judgement, planning and mental flexibility (Gouzoulis-Mayfrank et al., 1999Go). A recent study by Vollenweider showed definitively that sensory and cognitive effects of the hallucinogen psilocybin can be blocked by pre-administration of 5-HT2 receptor antagonists (Vollenweider et al., 1998Go). Both ketanserin and risperidone blocked, in a dose-dependent manner, psilocybin-induced psychosis, perceptual disturbances and spatial working memory deficits. Ketanserin is an antagonist for the 5-HT2 receptor that is 40 times more potent at blocking the 5-HT2A receptor than the 5-HT2C receptor (Roth et al., 1992Go). Risperidone is an atypical neuroleptic which has demonstrated 5-HT2A antagonism in addition to a lesser degree of D2 antagonism. In contrast, the typical neuroleptic haloperidol, which is a strong D2 antagonist, did not block the effect of psilocybin in healthy subjects.

In conclusion, there are profound laminar differences in the serotonergic modulation of glutamate transmission. This result is consistent with studies showing laminar differences in the density of the 5-HT2A receptor, in the density of 5-HT axons and in the density of projections from the midline thalamus. Many layer V pyramidal cells send projections to the basal ganglia, brainstem and spinal cord. Imaging and lesion studies suggest that there are thalamocortical-subcortical circuits which are critical for attention, motivation and ability to concentrate (Kinomura et al., 1996Go; Van Der Werf et al., 1999Go). Disrupting these circuits has been shown to have dramatic and deleterious effects on cognitive function. We have shown that 5-HT modulates neural transmission in one aspect of this circuit in vitro: the spontaneous release of glutamate onto layer V pyramidal cells. In vivo, animals placed in novel situations show increases in cortical 5-HT (Reuter and Jacobs, 1996Go) and 5-HT- dependent increases in motor activity (Geyer, 1996Go). Work from this laboratory suggests that these effects may be linked through the increase of asynchronous glutamatergic transmission (Aghajanian and Marek, 1999aGo) preferentially to the output neurons of the cerebral cortex. The potential role of 5-HT2A receptor stimulation as a marker of novelty is further supported by the rapid desensitization of the receptor under normal conditions (Aghajanian and Marek, 1997Go) and by the perturbations of cognition and behavior which result from the persistent activation of 5-HT2A receptors by psychedelic hallucinogens.


    Notes
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Notes
 References
 
This work was supported by National Institutes of Health Grant MH17871, the State of Connecticut and a predoctoral fellowship from the Bayer Foundation to E.K.L. We thank Nancy Margiotta for expert technical assistance. Address reprint requests to Dr George K. Aghajanian, Connecticut Mental Health Center, 34 Park St, New Haven, CT 06508, USA.

Address correspondence to Evelyn K. Lambe, Interdepartmental Neuroscience Program, Yale University School of Medicine, PO Box 208074, New Haven, CT 06520–8074, USA. Email: evelyn.lambe{at}yale.edu.


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 Discussion
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