Hippocampal 5-HT Input Regulates Memory Formation and Schaffer Collateral Excitation

Abstract

The efficacy and duration of memory storage is regulated by neuromodulatory transmitter actions. While the modulatory transmitter serotonin (5-HT) plays an important role in implicit forms of memory in the invertebrate Aplysia, its function in explicit memory mediated by the mammalian hippocampus is less clear. Specifically, the consequences elicited by the spatio-temporal gradient of endogenous 5-HT release are not known. Here we applied optogenetic techniques in mice to gain insight into this fundamental biological process. We find that activation of serotonergic terminals in the hippocampal CA1 region both potentiates excitatory transmission at CA3-to-CA1 synapses and enhances spatial memory. Conversely, optogenetic silencing of CA1 5-HT terminals inhibits spatial memory. We furthermore find that synaptic potentiation is mediated by 5-HT4 receptors and that systemic modulation of 5-HT4 receptor function can bidirectionally impact memory formation. Collectively, these data reveal powerful modulatory influence of serotonergic synaptic input on hippocampal function and memory formation.

Keywords: CA1; behavior; hippocampus; learning; memory; neuromodulation; optogenetics; plasticity; potentiation; serotonin.

Figure 1:. ChR2 expression in ePet1-cre;Ai32 mice.

(a) ChR2-YFP (green) is expressed throughout the raphe nucleus. DR- dorsal raphe, MR- medial raphe. Scale bar = 200 μm. (b, c) 94.8% of the cells analyzed co-stain for ChR2-YFP and 5-HT while 4.7% stain for 5-HT-only and 0.5% stain for ChR2-YFP-only. Scale bar = 50 μm (d) ChR2-YFP expression in the hippocampus. SR- stratum radiatum, SLM- stratum lacunosum moleculare, DG- dentate gyrus. Scale bar = 200 μm.

Figure 2:. ChR2-based optogenetic stimulation of serotonergic neurons using ePet1-cre;Ai32 mice.

Schematic for intracellular recordings (IC) in raphe containing brain slices of serotonergic neurons in the DR (a) and MR (e). Relationship between the frequency of stimulation using 473 nm pulses of light and the firing frequency of ChR2-YFP expressing DR (b) and MR (f) cells in acute brainstem slices. Vertical blue shading indicates optogenetic stimulation. Schematic for in vivo photostimulation of serotonergic neurons in the DR (c) and MR (g). ePet1-cre;Ai32 mice displayed hyperlocomotion when stimulated using blue light in the DR (d) or MR (h) (blue band; 473nm, 10ms, 20Hz). Control: ePet1-cre^−/−;Ai32^+/+; ChR2: ePet1-cre^+/−;Ai32^+/+. Posthoc genotype effect in three minute light on block is indicated; **: p < 0.01, ***: p < 0.001.

Figure 3:. Optogenetic activation of serotonergic fibers in CA1 elicits a fast transient hyperpolarization of CA1 pyramidal neurons.

(a) Experimental configuration and circuit schematic. (a-b) 2 or 20 Hz optogenetic stimulation of ePet1-cre^+/−;Ai32^+/− (ChR2^+/−) hippocampal slices induces a rapid hyperpolarization of CA1 pyramidal neurons. A cocktail of 5-HT receptor antagonists (GR113808, 100 nM; Odansetron, 10 nM; WAY100,635, 100 nM) blocked this hyperpolarization. Vm traces are taken from experiments representative of the population average. T-test based significant differences from baseline and between groups are indicated. *: p < 0.05, ***: p < 0.001.

Figure 4:. Optogenetic activation of serotonergic fibers in CA1 elicits a long-lasting potentiation of the CA3-CA1 synapse.

(a, d, g, k) Experimental configuration and circuit schematic. (a-c) When electrical stimulation of SR of CA1 at 0.05 Hz was paired with 50 blue light pulses (2 Hz), the PSP of CA1 pyramidal neurons increased. A cocktail of 5-HT receptor antagonists (GR113808, 100 nM; Odansetron, 10 nM; WAY100,635, 100 nM) blocked this potentiation of the CA3-CA1 synapse. (d-f) When the same protocol was applied to the perforant path we observed no potentiation. 2 Hz and 20 Hz experiments were averaged together because no effect of blue light stimulation frequency and no interaction between blue light stimulation frequency and time was detected. (g-i) When electrical stimulation of SR of CA1 at 0.05 Hz was paired with 50 blue light pulses (20 Hz), and local field potentials were recorded extracellularly (EC), the fEPSP in CA1 was increased. (k-m) When electrical stimulation of SR of CA1 at 0.2 Hz was paired with 50 blue light pulses (20 Hz), and local field potentials were recorded in vivo, the fEPSP in CA1 was increased. (b, e, h, l) Vm traces are taken from experiments representative of the population average. Pre and 10 min post optogenetic stimulation traces are overlaid. Vertical blue shading indicates optogenetic stimulation. (c, f, i, m) Scatterplots show the normalized PSP 10 – 15 minutes after optical stimulation (c, f), the normalized fEPSP 25 – 30 minutes after optical stimulation in slices (i), and the normalized fEPSP 20 – 25 minutes after optical stimulation in vivo (m). T-test based significant differences from baseline and between groups are indicated. *: p < 0.05, **: p < 0.01, ***: p < 0.001.

Figure 5:. Optogenetic activation of serotonergic terminals in CA1 increases water maze memory formation.

(a) Experimental design for stimulating CA1 5-HT release in vivo. No differences between genotypes and no effect of light stimulation was found on locomotion (b), time in the center in the OF (c), floating in the FST (d) or social recognition (e). Vertical blue shading indicates optogenetic activation of ChR2. (f-g) In the water maze, mice were trained for 5 days, 3 trials per day while stimulated with blue light at 20 Hz (blue dashes). (g) No effect of stimulation was detected during training. (h) During a 60s probe trial, ChR2-expressing mice spent more time in the zone where the platform was previously located (Target zone) than ChR2-non-expressing littermate control mice. The dashed line indicates random search. (i) ChR2 expressing mice crossed the area where the platform was previously located more often. (j) Density plots of probe trial behavior. Control: ePet1-cre^−/−;Ai32^+/+; ChR2: ePet1-cre^+/−;Ai32^+/+. *: p < 0.05, ***: p < 0.001.

Figure 6:. Optogenetic inhibition of serotonergic terminals in CA1 impairs water maze memory formation.

(a) Experimental design for inhibiting CA1 5-HT release in vivo. No effect of genotype or light was found on locomotion (b), time in the center in the OF (c), floating in the FST (d) or social recognition (e). Vertical green shading indicates optogenetic activation of Arch. (f-g) Mice were trained in the water maze for 6 days, 3 trials per day, given a first probe trial (P1), followed by 3 additional days of training and tested again in a second probe trial (P2), with green light administered as indicated (green dashes). (g) No effect of stimulation was detected during training. On P2 Arch expressing mice spent less time in the target zone (h) and crossed the platform area less times (i) than their non-expressing controls. (j) Density plots of P2 behavior. Control: ePet1-cre^−/−;Ai35^+/+; Arch: ePet1-cre+/−;Ai35^+/+. *: p < 0.05, **: p < 0.01, ***: p < 0.001.

Figure 7:. 5-HT4R-mediated modulation of hippocampal plasticity.

(a) 5-HT1AR antagonism (WAY100635, 100 nM) but not 5-HT4R antagonism (GR113808, 100 nM) or GABA receptor antagonism (SR-95531, 2 μM and CGP-35348, 1 μM) blocked the fast-transient hyperpolarization after optogenetic stimulation of serotonergic terminals in CA1. ChR2^+/−: ePet1-cre^+/−;Ai32^+/−. (b-c) 5-HT4R but not 5-HT1AR antagonism or GABA receptor antagonism blocked the potentiation of the CA3-CA1 synapse after optogenetic stimulation of serotonergic terminals in CA1 at either 2Hz or 20Hz. (c) Scatterplot shows the normalized PSP 10–15 minutes after optical stimulation. (d, e) Paired pulse ratio was not altered by optogenetic stimulation in either intracellular (d) or extracellular (e) slice recordings. (f) Input resistance (Rin) was increased after optogenetic stimulation. T-test based significant differences from baseline and between groups are indicated. *: p < 0.05, **: p < 0.01.

Figure 8:. Bidirectional modulation of water maze memory through pharmacological 5-HT4 receptor interference.

Mice were assessed in the MWM (a-h), OF (i-j) and FST (k). (a, b) Mice were treated with the 5-HT4 receptor antagonist GR125487 or with saline vehicle for 5 days, 20 min before water maze training. (c) During probe trial, no effect of treatment was detected for zone times, but (d) GR125487 injected mice crossed the platform area significantly fewer times. (e, f) Mice were treated with the 5-HT4 receptor agonist BIMU8 or with saline vehicle for 5 days, 20 min before water maze training. Probe trial performance was significantly improved in BIMU8 injected mice, which spent more time in the target zone on P2 (g) and crossed the platform location more often (h) than saline injected Controls. (i-k) Mice were treated with GR125487, BIMU8 or Saline 20 min before testing. BIMU8 treatment reduced ambulatory activity in the OF, while GR125487 had no effect (i). No effect of treatment was detected for OF center time (j) or FST floating time (k). *: p < 0.05, **: p < 0.01, ***: p < 0.001.