Regulation of Cortical 5-Hydroxytryptamine2A-Receptor Mediated Electrophysiological Responses in the Rat Following Daily Oral Lithium Administration

Research Article

Austin Med Sci. 2016; 1(1): 1005.

Regulation of Cortical 5-Hydroxytryptamine2A-Receptor Mediated Electrophysiological Responses in the Rat Following Daily Oral Lithium Administration

Marek GJ*

CNS & Pain, Astellas Pharma Global Development, USA

*Corresponding author: Marek GJ, CNS & Pain, Astellas Pharma Global Development, 1 Astellas Way, Northbrook, IL 60062, USA

Received: March 08, 2016; Accepted: May 09, 2016; Published: May 11, 2016


Down-regulation of 5-Hydroxytryptamine2A (5-HT2A) receptors has been a consistent effect induced by most antidepressant drugs. Lithium is the antimanic drug with the most comprehensive clinical literature in both unipolar depression and manic-depressive illness. With respect to unipolar depression, lithium is best established as an augmenting agent, but lithium also possesses modest antidepressant properties when used as monotherapy. The question of 5-HT2A receptor sensitivity during chronic antidepressant administration is important since activation of 5-HT2A receptors is associated with impulsivity and since down-regulation of 5-HT2A receptor binding/function has been associated with most antidepressant treatments. Therefore, the effects of subchronic oral lithium administration (one or two weeks) on pharmacologically characterized electrophysiological response mediated by 5-HT2A receptor activation in the Prefrontal Cortex (PFC) and the piriform cortex were examined. The concentration-response curve for 5-HT-induced EPSCs and 5-HT-induced firing of GABAergic interneurons recorded from the PFC and piriform cortical slices, respectively, was unchanged following subchronic lithium treatment. The efficacy of AMPA receptor activation was attenuated in the piriform cortex. While not having an effect on the concentration-response of 5-HT-induced EPSCs in the medial PFC, lithium did enhance both the onset and magnitude of desensitization for 5-HT-induced EPSCs in the PFC. Interestingly, lithium did not appear to attenuate the resensitization of 5-HT-induced EPCS. This asymmetric effect of lithium on 5-HT2A desensitization vs. resensitization in the PFC may be relevant for understanding the regulation of 5-HT2A receptor trafficking by lithium.

Keywords: Antidepressant drugs; 5-HT2A Receptors; Prefrontal cortex; Pyramidal cells; Interneurons; Mood stabilizers


5-HT2A: 5-hydroxytryptamine2A; ACSF: Artificial Cerebrospinal Fluid; AMPA: α-Amino-3-hydroxy-5-Methyl-4-isoxazolepropionate; Cg1: anterior cingulate cortex; Cg3: prelimbic cortex; DOI: 1(2,5-Dimethoxy-4-iodophenyl-2-aminopropane); EPSCs: Excitatory Postsynaptic Currents; GABA: γ−hydroxybutyric acid; mPFC: medial Prefrontal Cortex; PFC: Prefrontal Cortex; SSRIs: Selective Serotonin Reuptake Inhibitors


Lithium was the first medication established as a mood stabilizer for the treatment of manic-depressive illness [1]. Lithium also has a well-documented track record demonstrating efficacy in augmenting the antidepressant effect for a number of different antidepressant drug classes including tricyclic antidepressants, monoamine oxidase inhibitors, selective serotonin reuptake inhibitors, and atypical antidepressants such as mianserin [2-4]. Lithium also possesses modest, though frank antidepressant properties when used in monotherapy [1]. Furthermore, lithium also appears to reduce suicide risk [5], though this effect may be most robust when used in combination with antidepressant and/or antipsychotic medications [6-8]. A number of suggestions have been raised over the last 30 years to account for these robust clinical effects ranging from the phosphoinositide depletion hypothesis to alterations in neurotrophic signaling cascades [9-12].

One strategy emphasized by Manji and colleagues over the last decade has been to compare the effects of lithium with valproate and carbamazepine to triangulate upon downstream intracellular processes that all three agents share in common and that may account for the mood-stabilizing properties of these drugs in patients with bipolar disorder. However, lithium also possesses other actions described above for which valproate and carbamazepine have not been demonstrated to be active (e.g., augmentation of antidepressants in unipolar depression, frank antidepressant action in unipolar depression, and antisuicide effects).

The 5-HT2A receptor coupled to Gq/G11 and phospholipase C may be a link between therapeutically relevant pharmacological actions lithium and other known antidepressants. Involvement of lithium in the phosphatidyl inositol pathway, more specifically the “inositol sink” hypothesis was one of the earlier widely accepted proposals to explain at least some of the therapeutic effects of lithium [9,10]. Interaction of lithium with 5-HT2A receptors is of interest since most antidepressant drugs, outside the possible exception of fluoxetine and SSRIs, tend to decrease 5-HT2A receptor density and sensitivity in the PFC [13,14]. Furthermore, the post-mortem findings in suicidal depressed patients suggest that an up-regulation of 5-HT2A receptors may occur in certain regions such as Brodmann area 9 [15- 17]. Conversely, clozapine, which potently blocks 5-HT2A receptors in addition to other pharmacological effects, is known to decrease suicidality in schizophrenic patients [18]. Some authors have suggested that other atypical antipsychotics possessing potent 5-HT2A receptor blockade (e.g., olanzapine) may also have some degree of similar action [19,20].

Evidence for lithium-induced changes in 5-HT2A receptor binding and function in forebrain regions is mixed. For example two studies suggest that subchronic lithium treatment decreases 5-HT2A receptor binding in the rat PFC [21,22] while several other studies suggest that there is no change in the rat or mouse [23,24]. With regard to transduction pathways downstream from the receptor, lithium also failed to alter the 5-HT2A/2C receptor-mediated arachidonate response in most cortical regions outside of several sensory areas [25]. The 1-(2,5-Dimethoxy-4-iodophenyl-2-aminopropane (DOI)- induced c-fos induction in the cortex is increased by both acute and subchronic lithium administration, despite lack of evidence in this report for changes in 5-HT2A receptor binding with subchronic lithium treatment [24]. The most widely used behavioral model for 5-HT2A receptor function, DOI-induced head shakes, is not altered by subchronic lithium administration except for an interaction with TCAs in ACTH-treated rats [26]. Given these conflicting findings, it is important to understand whether subchronic lithium treatment modulates the electrophysiological effects of 5-HT2A receptor activation that by definition are generally measured in more homogeneous and discrete cellular compartments in different cortical regions such as the medial PFC and the piriform cortex.

Materials and Methods


Male Sprague-Dawley rats (n=51; Camm, Wayne, NJ) were 120- 200 g at the beginning of subchronic lithium treatment (approximately 5-7 weeks in age). All rats were provided a 7 day adaptation period following arrival from the supplier. They were housed in suspended stainless steel wireless cages (18 x 36 x 20 cm) with two rats occupying each cage. The colony room was maintained at 20°C and relative humidity (60%). The room was illuminated 12 h/day (0700-1900 hr). All rats had free access to laboratory chow (Teklad 4% Rat Diet) and regular drinking water in addition to hypertonic saline during the treatment phase. The principles of laboratory animal care (NIH publication No. 80-23, revised 1996) were followed. All procedures were approved by the Yale University Animal Care and Use Committee.

Lithium treatment

All subjects were treated with either normal drinking water and hypertonic saline during the treatment phase to prevent dehydration. All rats either continued on Teklad 4% Rat Diet (sham treatment) or were fed pellets containing 0.24% (wt/wt) lithium carbonate for 7, 14 or 28 days (including availability of hypertonic saline to prevent dehydration). The only exception to the above conditions was a portion of subjects in the 5-HT2A receptor desensitization experiment which were naïve to treatment (n=4), but were tested concurrently with the rats administered either the lithium-containing pellets or the sham diet treatment. An additional cohort of 13 rats were treated with lithium via the lithium-containing pellets or the sham treatment for 1 week prior to examining desensitization of 5-HT-induced EPSCs in the mPFC (n= 7, lithium; n=6, control). Overall, 29 rats were treated for 1, 2 or 4 weeks with lithium and compared with 35 control rats. Male Sprague-Dawley rats previously treated for 6 days to 4 weeks [27] have been found to have serum lithium levels within the target range for the acute treatment of bipolar patients (0.98 + 0.18 and 1.1 + 0.25 mM, respectively).

Brain slice preparation

Brain slices were prepared as described previously [14]. Briefly, rats were anesthetized with chloral hydrate (400 mg/kg, i.p.) and decapitated. Coronal slices (500 μM) were cut with an oscillatingblade tissue slicer at a level corresponding to approximately 2.5 mm anterior to bregma for recording from the medial Prefrontal Cortex (mPFC) or to approximately 2.2 mm anterior to bregma for recording from the anterior piriform cortex. A slice containing the mPFC or piriform cortex was then transferred to the stage of a fluidgas interface chamber, which had a constant flow of humidified 95% O2, 5% C02. The slices were perfused in a chamber heated to 34°C with normal ACSF, which consisted of 126 mMNaCl, 3 mMKCl, 2 mM CaCl2, 2 mM MgSO4, 26 mM NaHC03, 1.25 mM NaH2P04, and 10 mM D-glucose.

Electrophysiological recording

Intracellular recording and single-electrode voltage clamping were conducted in prefrontal cortical layer V pyramidal cells by using an Axoclamp-2A (Axon Instruments, Inc., Foster City, CA) as previously described [14,28]. Stubby electrodes (~8 μm, shank to tip) with relatively low capacitance and resistance (30-60 MOhms) were filled with 1M potassium acetate. The cells were voltage clamped at -70 mV. Phase lag was used to prevent oscillations; false clamping was avoided by utilizing optimal capacitance neutralization and by allowing settling to a horizontal baseline, as verified by monitoring input voltage continuously. Layer V pyramidal cells were recorded in a zone one-half to two-thirds the distance between the pial surface and the white matter in the mPFC (anterior cingulate and prelimbic area; Cg1, Cg3). The Excitatory Postsynaptic Currents (EPSCs) recorded under these conditions do not appear to be contaminated by reversed inhibitory postsynaptic currents as previously discussed [28]. The voltage-clamp signals were low-pass filtered (1000 Hz) and data were acquired with a pCLAMP/Digidata 1200 system (Axon Instruments, Inc., Foster City, CA).

Extracellular recordings were conducted using an Axoclamp- 2A (Axon Instruments, Burlingame, CA, USA) [29]. Placement of the recording electrodes was guided by visualizing the three layers of the piriform cortex at low magnification under reflected light. Extracellular recordings from interneurons located in layer III of the piriform cortex were made using glass microelectrodes filled with 2 M NaCl (5-10 MOhms) as described previously [29]. Cells were found by searching while the slice was perfused with 30 μM 5-HT in order to activate quiescent cells. 5-HT was applied for no longer than 15 min and was turned off for an equivalent period if necessary to search for an additional cell. Cells identified as interneurons had the ability to sustain rapid firing rates as previously characterized by both intracellular and extracellular recording [29,30]. Once an interneuron was located, the solution was switched from 30 μM 5-HT back to control ACSF and the 5-HT activated cells gradually slowed and, in almost all cases, ceased firing. Then the effects of 5-HT creatine sulfate (Sigma Chemical Co., St. Louis, MO, USA) and α amino-3-hydroxy- 5-methyl-isoxazoleproprionate (AMPA; RBI, Natick, MA, USA) were tested. It should be noted that continuous 5-HT application has not been found to produce significant tachyphylaxis (>10-15%) of the 5-HT-induced firing rate in the piriform cortex either when measured during continuous application or when measuring the steady-state response to 30 μM 5-HT with 15 min durations between consecutive applications (not shown).

Data analysis

For the intracellular recordings, EPSC frequencies were obtained from 10 consecutive episodes (1 s duration) during the baseline and drug treatment periods. EPSC frequencies were determined with Axograph peak detect software; signals <10 pA were excluded from the measurements. The determination of EC50 values for the suppression of 5-HT-induced increases in EPSC frequency were calculated by nonlinear curve fitting (Graphpad Prism; For the extracellular recordings, the total number of spikes for each concentration of 5-HT (10, 30, 100 and 300 μM) was recorded. A twofactor (5-HT concentration and drug treatment) repeated measures ANOVA was performed. Post-hoc follow-up of significant results utilized the Newman-Keuls test. Comparisons for treatment effects on AMPA-mediated responses utilized a paired t-test. Significance levels was set at p<0.05.


Subchronic lithium: Piriform cortical interneurons

A two-week exposure to lithium carbonate, in concentrations previously demonstrated to alter downsteam intracellular transduction pathways for surface G-protein coupled receptors, failed to result in a significant effect for the lithium exposure vs. sham treatment (F(1,16)=1.81, p=0.197; Figure 1) when examining 5-HTinduced activation of GABAergic interneurons of the piriform cortex (n=8 lithium treated rats; n=10 control rats). An expected highly significant effect of 5-HT concentration was found (F(3,48)=175.57, p<0.001). The interaction of these two factors was not significant (F(3, 48)=0.933, p=0.43). In a subset of these cells, subchronic lithium exposure did attenuate the response of piriform interneurons to bath application of AMPA (5 μM; t(7)=2.82, p<0.05; Figure 1; lithium n=6; control n=3). Extending the lithium exposure to a four week period did not appear to alter the 5-HT concentration-response curve in limited set of cells (n=4, lithium; n=3, sham treatment, not shown).