The impact of pitolisant, an H3 receptor antagonist/inverse agonist, on perirhinal cortex activity in individual neuron and neuronal population levels

Abstract

Histamine is a neurotransmitter that modulates neuronal activity and regulates various brain functions. Histamine H₃ receptor (H₃R) antagonists/inverse agonists enhance its release in most brain regions, including the cerebral cortex, which improves learning and memory and exerts an antiepileptic effect. However, the mechanism underlying the effect of H₃R antagonists/inverse agonists on cortical neuronal activity in vivo remains unclear. Here, we show the mechanism by which pitolisant, an H₃R antagonist/inverse agonist, influenced perirhinal cortex (PRh) activity in individual neuron and neuronal population levels. We monitored neuronal activity in the PRh of freely moving mice using in vivo Ca²⁺ imaging through a miniaturized one-photon microscope. Pitolisant increased the activity of some PRh neurons while decreasing the activity of others without affecting the mean neuronal activity across neurons. Moreover, it increases neuron pairs with synchronous activity in excitatory-responsive neuronal populations. Furthermore, machine learning analysis revealed that pitolisant altered the neuronal population activity. The changes in the population activity were dependent on the neurons that were excited and inhibited by pitolisant treatment. These findings indicate that pitolisant influences the activity of a subset of PRh neurons by increasing the synchronous activity and modifying the population activity.

Figure 1

In vivo Ca²⁺ imaging from perirhinal cortex neurons. (A) GRIN lens is implanted above the PRh, and images are acquired using the GRIN lens and miniscope. (B) A representative image depicting the GRIN lens position and GCaMP6m expression in PRh neurons. (C,D) Representative correlation images (left) and extracted Ca²⁺ traces before and after saline (C) or pitolisant (D) injection. DLEnt, dorsolateral entorhinal cortex; GRIN, gradient-index; and PRh, perirhinal cortex.

Figure 2

Pitolisant substantially alters the activity of a subset of neurons. (A) A proportion of neurons displaying excitatory, inhibitory, or stable responses to saline or pitolisant treatment (saline: 184 neurons, pitolisant: 178 neurons). (B) Neurons displaying an excitatory response to pitolisant treatment have higher activity scores, compared with saline treatment (*p = 0.022, Mann–Whitney U test; saline, 35 neurons; pitolisant, 32 neurons). (C) Neurons displaying an inhibitory response to pitolisant treatment have lower activity scores, compared with saline treatment (**p = 0.0014, Mann–Whitney U test; saline, 50 neurons; pitolisant, 43 neurons). (D) Activity scores of stable neurons are comparable between saline and pitolisant treatments (p = 0.5448, Mann–Whitney U test; saline, 99 neurons; pitolisant, 103 neurons).

Figure 3

Calcium event frequency from excitatory and inhibitory responsive neurons differ between saline and pitolisant treatments. (A) The event frequency score of excitatory responsive neurons to pitolisant treatment is higher compared with saline treatment (*p = 0.029, Mann–Whitney U test). (B) The event frequency score of inhibitory responsive neurons to pitolisant treatment is lower compared with saline treatment (**p = 0.0030, Mann–Whitney U test). (C) The size of calcium events following pitolisant treatment is larger compared with saline treatment (*p = 0.021, Mann–Whitney U test). (D) The size of calcium events from inhibitory responsive neurons is comparable between saline and pitolisant treatments.

Figure 4

Pitolisant enhances the proportion of synchronously active neuron pairs within excitatory responsive neuronal populations. (A) Proportions of cell pairs with synchronous activity do not differ between neuronal populations displaying excitatory and stable responses to saline treatment (p = 0.81, Fisher's exact test). (B) More cell pairs display synchronous activity within excitatory responsive neuronal populations to pitolisant treatment, compared with stable neuronal populations (**p = 6.2 × 10^–5, Fisher's exact test).

Figure 5

Population activity before and after pitolisant treatment is accurately classified by linear support vector machine. (A) Accuracy of two-way decoders discriminating before and after treatment. Decoders based on population activity data before and after pitolisant treatment performed better, compared with saline treatment (**p = 0.0053, paired t-test). (B) Decoders constructed from single neuron data before and after pitolisant treatment performed poorer, compared with saline treatment (*p = 0.045, paired t-test). (C) Decoders constructed from population activity data without excitatory responsive neurons performed similarly between saline and pitolisant treatments (p = 0.3020, paired t-test). (D) Decoders constructed from population activity data without inhibitory responsive neurons performed similarly between saline and pitolisant treatments (p = 0.0897, paired t-test). (E) Decoders constructed from population activity data without stable neurons performed better than those treated with saline (**p = 0.0038, paired t-test). Data are reported as mean ± SEM.