This chapter (2017) reviews the effects of serotonergic psychedelics on cortical layer V pyramidal neurons.
Abstract
“Recordings made from layer V (L5) pyramidal cells of the prefrontal cortex (PFC) and neocortex in rodent slice preparations have shown that serotonin (5-hydroxytryptamine, 5-HT) and serotonergic hallucinogens induce an increase in the frequency of spontaneous excitatory postsynaptic currents (EPSCs) in the apical dendritic field by activating 5-HT2A receptors. Serotonergic hallucinogens induce late EPSCs and increase recurrent network activity when subcortical or mid-cortical regions are stimulated at low frequencies (e.g., 0.1 Hz). A range of agonists or positive allosteric modulators (PAMs) for mostly Gi/o-coupled receptors, including metabotropic glutamate2 (mGlu2), adenosine A1, or μ-opioid receptors, suppress these effects of 5-HT2A receptor stimulation. Furthermore, a range of mostly Gq/11-coupled receptors (including orexin2 [OX2]; α1-adrenergic, and mGlu5 receptors) similarly induce glutamate (Glu) release onto L5 pyramidal cells. Evidence implicates a number of brain regions in mediating these effects of serotonergic hallucinogens and Gq/11-coupled receptors including the midline and intralaminar thalamic nuclei, claustrum, and neurons in deep PFC. These effects on 5-HT2A receptors and related GPCRs appear to play a major role in the behavioral effects of serotonergic hallucinogens, such as head twitches in rodents and higher order behaviors such as rodent lever pressing on the differential-reinforcement-of-low rate 72-s (DRL 72-s) schedule. This implies that the effects of 5-HT2A receptor activation on the activity of L5 pyramidal cells may be responsible for mediating a range of behaviors linked to limbic circuitry with connectivity between the PFC, striatum, thalamus, claustrum, striatum, amygdala, and the hippocampal formation.”
Author: Gerard J. Marek
Summary
Interactions of Hallucinogens with the Glutamatergic System: Permissive Network Effects Mediated Through Cortical Layer V Pyramidal Neurons
Serotonin and serotonergic hallucinogens activate 5-HT2A receptors in layer V pyramidal cells of the prefrontal cortex and neocortex, which are responsible for mediating behaviors such as head twitches and lever pressing on the differential-reinforcement-of-low rate 72-s (DRL 72-s) schedule.
The present chapter will discuss evidence demonstrating that hallucinogenic drugs stimulate cortical 5-HT2A receptors, and may provide an important clue to the treatment of psychotic disorders. The genesis of this line of work began with the discovery of LSD and the observation that it mimics certain symptoms exhibited by patients with schizophrenia. Later, studies suggested that LSD, mescaline, and psilocybin all share a common agonist action at 5-HT2A receptors.
In 1997, it was discovered that serotonin induces spontaneous excitatory postsynaptic potentials in prefrontal cortical and neocortical layer V (L5) pyramidal neurons in rat brain slice preparations. These EPSPs were potently blocked by 5-HT2A receptor antagonists.
5-HT-induced EPSPs/EPSCs were suppressed by the AMPA receptor antagonist LY293558, the GABAA receptor antagonist bicuculline, the fast sodium channel blocker tetrodotoxin, and the iontophoretic application of 5-HT in layers I and Va in a distribution consistent with stimulation of the apical dendrite but not the basilar dendrite. 5-HT releases Glu from glutamatergic afferents terminating in the layer I and Va dendritic field of L5 pyramidal cells, which in turn excite principle output neurons of the prefrontal cortex and neocortex.
A model paradigm for observing Glu release induced by serotonergic hallucinogens via 5-HT2A receptor activation in prefrontal cortical slices was subsequently identified. This model involves applying a serotonergic hallucinogen for 10 min, followed by focal electrical stimulation of the slice at 1 Hz. 5-HT1A, 5-HT1B, and 5-HT1F receptors are inhibited by 5-HT in the PFC, whereas 5-HT2A receptors are activated by 5-HT. This results in excitatory feedforward and feedback recurrent network activity.
The 5-HT-induced EPSCs and recurrent network activity induced by the combination of DOI and focal low-frequency electrical stimulation are suppressed by a range of neurotransmitter receptors that are known to inhibit presynaptic Glu release. The mGlu2/3 receptors may play an important role in modulating the Glu release induced by 5-HT2A receptor activation. In the rat PFC, mGlu2 receptors act as an autoreceptor on glutamatergic terminals releasing Glu (induced by 5-HT2A receptor activation) from afferents reaching the layer I and Va dendritic field of L5 pyramidal cells.
Several G-protein-coupled receptors, including mGlu4 and mGlu8, were found to suppress spontaneous 5-HT-induced EPSCs, as were adenosine A1 receptors and the l-opioid receptor. Thalamic lesions were also found to decrease mGlu2/3 receptor binding sites, suggesting a potential source for the 5-HT-induced EPSCs. The localization of mGlu2,mGlu4, adenosine A1, and l-opioid receptors in the midline and intralaminar thalamic nuclei is consistent with a model where 5-HT2A receptor activation and a range of Gi/o-coupled GPCRs have countervailing effects Glu release.
Several Gs-coupled GPCRs appear to have similar effects on hallucinogen-induced recurrent network activity, including the D1/D5 dopamine receptors, which are coupled to Gs. The effects of forskolin and 8-Br-cAMP on DOI-induced recurrent network activity occurred in the absence of effects on fast early EPSCs. The D1/D5 ligands have a cortical versus thalamic site of action and are supported by a range of known mechanisms for dopamine D1/D5 receptors.
A study suggested that b2-adrenergic receptor stimulation by epinephrine or clenbuterol can suppress DOI-induced recurrent network activity, and that this effect is also consistent with a role of thalamic inputs in DOI-induced Glu release in the mPFC.
Activation of a number of Gq/11-coupled GPCRs induces feedforward Glu release onto L5 pyramidal cells in the PFC and/or neocortex, similar to the effect of 5-HT via 5-HT2A receptors.
NE-induced spontaneous EPSCs are especially interesting because a1-adrenoceptors and 5-HT2A receptors have significant similarities with respect to regional brain distribution, laminar cortical protein distribution, and laminar cortical mRNA distribution, as well as a range of physiological and behavioral effects with salient medial PFC involvement.
In rat brain slices, the type I mGlu receptor agonist DHPG induces spontaneous EPSCs that can be blocked by a selective negative allosteric modulator of the mGlu5 receptor. In addition, a l-opioid receptor agonist and a AMPA/GluK5 receptor antagonist suppress DHPG-induced EPSCs. The pharmacology of senktide-induced increase in EPSC frequency has not been defined beyond blockade by NK3 receptor antagonists. However, studies in rats suggest that NK3 receptors are present in the midline thalamic nuclei.
Acetylcholine and nicotine were found to induce EPSCs/EPSPs through activation of a4b2 nicotinic receptors on thalamic afferents to L5 pyramidal cells. ACh may also increase the frequency of spontaneous EPSCs.
At least a majority of 5-HT-induced EPSCs appear to originate from subcortical sources, and fiber-sparing chemical or radiofrequency lesions of the midline and intralaminar thalamic nuclei reduce the frequency of 5-HT-induced EPSCs in rat PFC slices. Postsynaptic 5-HT2A receptors were significantly upregulated in response to chemical fiber-sparing thalamic lesions, but presynaptic 5-HT2A receptors were eliminated, suggesting that glutamatergic terminals and axons expressing 5-HT2A receptors are relatively rare in the mPFC. A subsequent report confirmed that 5-HT induces EPSCs in the PFC via closing Kv1.2-containing potassium channels, and that thalamic lesions reduce the frequency of nicotine- and ACh-induced spontaneous EPSCs by 80%.
Around this time, evidence emerged that activation of orexin2 (OX2) receptors induced spontaneous EPSCs via activation of afferents from the midline and intralaminar thalamic nuclei. The effects of TTX, l-opioid agonists, and an AMPA receptor antagonist were similar to those observed previously for 5-HT2A receptor activation.
The partial suppression of 5-HT-induced EPSCs in the mPFC by large thalamic lesions suggests that there is at least one additional source of afferents to the L5 pyramidal cells.
Several cortical sources have been proposed to mediate the EPSCs induced by 5-HT and serotonergic hallucinogens. These sources include deep cortical neurons that are depolarized sufficiently by 5-HT2A receptor activation to fire action potentials, and L5 pyramidal neurons that send callosal and commissural projections to the contralateral cortex.
One report suggested that 5-HT-induced EPSCs arise primarily from neocortical afferents intrinsic to the cerebral cortical mantle. However, interpretation of the htr2a gene rescue experiment for the thalamus appears to have been compromised.
The spontaneous EPSCs induced by 5-HT and hypocretin-2 are likely mediated by afferents arising from the midline and intralaminar thalamic nuclei. However, further work is necessary to understand the role of claustral and/or neocortical afferents in mediating the effects of hallucinogenic drugs.
1 From 5-HT2AReceptor-Induced EPSCs and RecurrentNeuronal Activity to Salient Behavioral Effects Induced by 5-HT2AReceptor Activation
Head-Twitch Response
The behavioral effects of hallucinogenic drugs are modulated by the same neurotransmitter systems that affect Glu released from subcortical terminals and intrinsic cortical afferents onto the apical dendrites of L5 pyramidal cells.
According to a report published almost 20 years ago, hallucinogen-induced head twitches are mediated by activation of 5-HT2A receptors in the mPFC. Several antidepressant drugs, typical and atypical antipsychotic drugs, and mGlu2/3 receptor agonists were found to suppress the HTR induced by DOI, 5-HTP, and quipazine, whereas the mGlu2/3 receptor antagonist LY341495 potently enhanced the frequency of DOI-induced head twitches. A range of neurotransmitter receptor agonists, PAMs, and antagonists that modulate 5-HT2A receptor activation have been found to suppress head twitches induced by serotonergic hallucinogens in rodents.
Dopamine D1/5 receptor antagonists were found to suppress, rather than enhance, DOI-induced head twitches, but the mechanism responsible for this effect is unclear. Nicotine is another ligand that has been tested and found to have effects opposite to those expected.
DOI and the NMDA receptor antagonist MK-801 have synergistic effects on horizontal locomotor activity in rats, consistent with the hypothesis that modulation of Glu spillover is a key substrate for modulating the HTR in mice and rats, a well-known behavioral effect of serotonergic hallucinogens.
Motoric impulsivity studied using the 5-CSRTT
The 5-choice serial reaction time test (5-CSRTT) is an intriguing behavioral paradigm that has been studied with respect to activation of 5-HT2A receptors in the PFC. 5-HT2A receptor antagonists reduce motoric impulsivity in 5-CSRTT studies, but do not alter another form of impulsivity related to delayed reward presentation.
The 5-CSRTT has consistent behavioral evidence for a modulatory effect of dopamine D1/5 receptors, although the effect of DOI on premature responding has been reported to be suppressed by a dopamine D1 receptor antagonist.
Rats operating under a differential-reinforcement-of-low rate 72-sec schedule of reinforcement are biased towards responding at time durations matching reinforcement contingencies, and antidepressant drugs as a class appear to induce cohesive antidepressant-like rightward shifts in the inter-response time distribution.
Blockade of NMDA receptors and adenosine A1 receptors results in antidepressant-like effects in rats responding under DRL 72-s schedules. Clenbuterol and other b2-adrenergic receptor agonists do not appear to induce antidepressant-like effects on DRL 72-s behavior.
The lack of antidepressant-like effects on DRL behavior for some agents known to suppress Glu release onto L5 pyramidal cells is not surprising given that these agents also possess antidepressant-like or antipsychotic-like behavioral effects.
In vitro colocalization of 5-HT2A and mGlu2 receptors and a wealth of functional interactions have led to the suggestion that 5-HT2A and mGlu2 receptors form a heterocomplex in L5 pyramidal cells in the PFC and neocortex. However, functional evidence for heterocomplexes could not be confirmed in in vitro experiments. Doumazane and colleagues did produce evidence for heterodimers between mGlu2 and mGlu4 receptors in overexpressing cell lines, but this evidence ignores the data suggesting that only a very minor subpopulation of 5-HT2A receptors are expressed presynaptically and the overwhelming evidence for a predominantly presynaptic function of the mGlu2 receptor. Mutating three residues from the intracellular portion of the mGlu2 receptor suppresses 5-HT2A receptor activity, which strongly supports the existence of 5-HT2A- and mGlu2-containing heterocomplexes.
D9-tetrahydrocannabinol (THC)-induced amnesia appears to be caused by heteromers between 5-HT2A receptors and cannabinoid CB1 receptors, which share some similarity to 5-HT2A – mGlu2 heterocomplexes.
2 Clinical Relevance of the Interactions Between Glutamate and 5-HT2A Receptors in the mPFC
Several 5-HT2A receptor antagonists were discovered, characterized, and tested in patients with schizophrenia. The results were disappointing, with none of the drugs exerting antipsychotic effects approaching those of either first- or second-generation antipsychotic drugs.
Pimavanserin, a selective 5-HT2A receptor antagonist or inverse agonist, has been shown to improve psychosis in patients with Parkinson’s disease. This suggests that 5-HT2A receptor blockade may provide clinically relevant therapeutic effects for psychosis in patients with neurodegenerative disease.
A phase 2 trial addressing the effects of pimavanserin on psychosis associated with Alzheimer’s disease (AD) was recently completed and reportedly showed positive results. This study is intriguing given that down-regulation of 5-HT2A receptors occurs in AD before other neurotransmitter receptors are altered, and shared pathophysiology exists between AD and PD.
Recent studies suggest that 5-HT2A receptor inverse agonists can be used to treat psychosis associated with Parkinson’s disease and Alzheimer’s disease, and that mGlu2/3 receptor agonists may have a larger effect size in these patients.
A number of drugs targeting receptor sites that modulate 5-HT-induced EPSCs in rat mPFC slice preparations also exert antidepressant-like effects in the DRL 72-s schedule. These findings are consistent with double-blind, placebo-controlled studies suggesting antidepressant efficacy for drugs such as trazodone, nefazodone, mirtazapine, and mianserin. Previous studies suggested that b2-adrenergic receptor agonist salbutamol might have antidepressant effects in depressed patients, but a mGlu2 receptor PAM is the only novel mechanism suppressing 5-HT-induced EPSCs that has also been tested in a clinical study. A number of open-label trials with buprenorphine in patients with MDD have suggested that this compound has antidepressant activity, consistent with opiates’ long history of use in depressed patients. A selective OX2 receptor antagonist is currently being developed by Minerva Neurosciences and Janssen for the treatment of insomnia and MDD. Preliminary reports indicate that there were improvements in both sleep and mood in patients with MDD.
Ketamine, a channel blocking NMDA receptor antagonist, induces new synaptic spines in L5 pyramidal cells within 24 h of administration, suggesting that this drug may be useful in treating treatment refractory MDD or bipolar depression. Ketamine exerts antidepressant-like effects in the forced swim test, the learned helplessness paradigm, and the novelty suppressed feeding test 24 h after administration. Ketamine also reverses deficits in synaptic electrophysiology, spine number, synaptic protein expression, and behavior induced by 21 days of chronic stress exposure.
Recent findings indicate that lithium potentiates the electophysiological, synaptogenic, and antidepressant-like behavioral effects of ketamine in rats, and that scopolamine produces similar effects in preclinical rodent studies that parallel its clinical antidepressant effects observed for MDD and bipolar disorder, including treatment refractory cases.
3 Conclusions
Serotonergic hallucinogens such as LSD, mescaline, and psilocybin have inspired research goals ranging from curing major neuropsychiatric diseases to understanding consciousness. A number of monoaminergic, glutamatergic, purinergic, and peptide neurotransmitter receptor ligands suppress Glu release from putative thalamic afferents and from other sources, and suppress the HTR induced by serotonergic hallucinogens. These drugs also have antidepressant-like and/or antipsychotic-like effects in patients. Recent studies suggest that unique electrophysiological, synaptogenic, and behavioral profiles may help uncover new medications for treatment refractory MDD and bipolar depression.