Regularizing firing patterns of rat subthalamic neurons ameliorates parkinsonian motor deficits

Abstract

The subthalamic nucleus (STN) is an effective therapeutic target for deep brain stimulation (DBS) for Parkinson's disease (PD), and histamine levels are elevated in the basal ganglia in PD patients. However, the effect of endogenous histaminergic modulation on STN neuronal activities and the neuronal mechanism underlying STN-DBS are unknown. Here, we report that STN neuronal firing patterns are more crucial than firing rates for motor control. Histamine excited STN neurons, but paradoxically ameliorated parkinsonian motor deficits, which we attributed to regularizing firing patterns of STN neurons via the hyperpolarization-activated cyclic nucleotide-gated channel 2 (HCN2) channel coupled to the H2 receptor. Intriguingly, DBS increased histamine release in the STN and regularized STN neuronal firing patterns under parkinsonian conditions. HCN2 contributed to the DBS-induced regularization of neuronal firing patterns, suppression of excessive β oscillations, and alleviation of motor deficits in PD. The results reveal an indispensable role for regularizing STN neuronal firing patterns in amelioration of parkinsonian motor dysfunction and a functional compensation for histamine in parkinsonian basal ganglia circuitry. The findings provide insights into mechanisms of STN-DBS as well as potential therapeutic targets and STN-DBS strategies for PD.

Keywords: Neuroscience; Parkinson’s disease.

Figure 1. Histaminergic afferents in the STN and the histamine-induced amelioration of turning behavior of PD rats.

(A) Sagittal view of the rat brain showing the localization of the STN between –3.60 and –4.30 mm from the bregma. STN tissue punches for analysis of histamine levels were collected from brain slices obtained using these coordinates. (B) HPLC and ELISA analyses show levels of histamine (ng/g of tissue) in the ipsilesional and contralesional STN of PD rats (n = 10) on 1, 7, 14, and 21 days after 6-OHDA injection (n = 5). (C) Immunofluorescence staining shows that anterogradely labeled BDA fibers in the STN, originating from the histaminergic neurons in the hypothalamic TMN (left panels), contained histamine immunoreactivity (right panels). Note that these histaminergic fibers possessed prominent varicosities (indicated by arrows) and passed around (indicated by arrowheads) glutamate immunoreactive (glutamatergic) neurons in the STN (3 independent experiments). cp, cerebral peduncle; ic, internal capsule; LV, lateral ventricle; ZI, zona incerta. (D) Behavioral tests show that histamine (1 μg) microinjected into STN decreased, whereas high K⁺ (0.75 μg KCl) increased, the rate and total number of apomorphine-induced turnings in 30 minutes in PD rats (n = 12). Data are represented as mean ± SEM or median (horizontal bar) with 25th–75th (box) and 5th–95th (whiskers) percentiles. *P < 0.05; ***P < 0.001, 2-way (B) or 1-way ANOVA (D) with Newman-Keuls post hoc test.

Figure 2. The histamine-induced regularization of firing patterns of STN neurons in normal and PD rats.

(A–F) Effects of histamine (1 μg) and high K⁺ (0.75 μg KCl) on firing rate and firing pattern of 2 recorded STN neurons in normal and PD rats in vivo. PSTHs (A and D) show that both histamine and high K⁺ excited the STN neuron. Insets represent 5 superimposed traces of spike waveforms for each unit, respectively. Autocorrelation histograms (B and E) show that histamine, rather than high K⁺, promoted periodicity of STN neuronal firing. ISI histograms (C and F) show that histamine, but not high K⁺, narrowed ISI distributions. (G–I) Histamine increased firing rates (G, n = 30), decreased CV of ISIs (H, n = 15), reduced number of bursts (I, left panel, n = 15), and prolonged the interburst intervals (I, right panel, n = 15) of STN neurons in both normal and PD rats. However, high K⁺ only increased firing rates (G), but did not influence firing patterns (H and I) of STN neurons in normal and PD rats. Data are represented as mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001, 2-way ANOVA with Newman-Keuls post hoc test (G–I).

Figure 3. HCN channel coupled to H2 receptor mediates the effect of histamine on STN neurons in normal rats.

(A) Microscope image of a STN neuron (indicated by arrowheads) recorded in a brain slice and labeled with biocytin after patch-clamp recording. (B) Histamine (10 μM) shifted the conductance-voltage curve of recorded STN neurons (at –90 mV and –100 mV, n = 5). The conductance curve was converted from the whole-cell currents recorded from –50 to –120 mV and was fitted by Boltzmann function. Note that the conductance exhibited a significant feature of hyperpolarization activation and histamine reduced the voltage required for half-maximal activation (V_1/2, n = 5). (C) Histamine increased the inward rectification (sag) in response to an 80 pA hyperpolarizing current pulse. ZD7288 (50 μM), a selective blocker for the HCN channel, abolished the depolarizing sag in both the absence and presence of histamine (n = 8). (D) Histamine elicited an inward current in a STN neuron, and ranitidine (1 μM), a selective antagonist for the H2 receptor, or ZD7288 (50 μM) totally blocked the current induced by histamine (n = 8). (E and F) PSTHs, scatter plots of ISI series, autocorrelation histograms, and an ISI histogram of discharges of a recorded STN neuron show the histamine-induced changes in firing rate and firing pattern in the absence and presence of ranitidine and ZD7288 (1 μM, respectively). (G) Group data show that histamine significantly decreased the CV of ISIs, whereas ZD7288 remarkably increased the CV and blocked the histamine-induced decrease in CV (n = 30). Data are represented as mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001, 2-tailed paired t test (B) or 1-way ANOVA with Newman-Keuls post hoc test (C, D, and G).

Figure 4. HCN channel coupled to H2 receptor mediates the histamine-induced amelioration of motor deficits in PD rats.

Effects of microinjection of histamine (1 μg), dimaprit (a selective agonist for H2 receptor, 2 μg), ranitidine (a selective antagonist for H2 receptor, 3.5 μg), and ZD7288 (a selective blocker for HCN channel, 3 μg) into the STN on turning behavior (A, n = 12), adhesive-removal test (B, n = 10), and locomotor footprints (C, n = 10) in PD rats. Dimaprit mimicked the histamine-induced decrease in turnings, shortened time for removing an adhesive strip from forelimb contralateral to the lesion, and enlarged bilateral stride lengths, whereas ranitidine and ZD7288 significantly increased the turnings, prolonged contralesional adhesive-removal time, and shortened bilateral stride lengths. ZD7288 also abolished the histamine-induced amelioration of turning, adhesive removal, and locomotor behaviors in PD rats. Data are represented as median (horizontal bar) with 25th–75th (box) and 5th–95th (whiskers) percentiles or mean ± SEM. **P < 0.01; ***P < 0.001, 1-way (A and C, stride width) or 2-way ANOVA (B and C, stride length) with Newman-Keuls post hoc test.

Figure 5. The HCN2 channel is responsible for the histamine-induced amelioration of motor deficits in PD rats.

(A–D) LV-Hcn1-shRNA, LV-Hcn2-shRNA, LV-Hcn3-shRNA, and LV-Hcn4-shRNA effectively downregulated the expression of Hcn1, Hcn2, Hcn3, and Hcn4 mRNAs and proteins (n = 6 from 6 independent experiments) in the STN. (E) LV-Hcn2-oe upregulated the expression of Hcn2 mRNAs and proteins (n = 6 from 6 independent experiments). (F–H) Effects of downregulation and overexpression of the HCN2 channel in the STN on motor deficits of turning behavior (F, n = 12), adhesive-removal test (G, n = 10), and locomotor footprints (H, n = 10) in PD rats with sham operation, saline injection, and histamine injection. Downregulation of the HCN2 channel significantly increased the apomorphine-induced turnings, prolonged contralesional adhesive-removal time, and shortened bilateral stride length, whereas downregulation of the HCN1, HCN3, or HCN4 channel had no effect on these motor deficits. Only the downregulation of HCN2 rather than the HCN1, HCN3, or HCN4 channel blocked the amelioration of turnings, removal time, and stride length of PD rats induced by microinjection of histamine into the STN. Overexpression of the HCN2 channel in STN not only decreased the turnings, reduced removal time, and enlarged bilateral stride length of PD rats, but also improved the histamine-induced amelioration in these motor behaviors. Data are represented as mean ± SEM. ***P < 0.001, 1-way (A–E) or 2-way ANOVA (F–H) with Newman-Keuls post hoc test.

Figure 6. Overexpression of the HCN2 channel, as well as STN-DBS, regularizes firing patterns of STN neurons in PD rats.

(A) Three continuous oscilloscope traces show the firings of 3 STN neurons in a PD rat, a PD rat with overexpression of HCN2 in STN, and a PD rat with downregulation of HCN2. Insets represent 5 superimposed traces of spike waveforms for each unit. (B–D) Autocorrelation histograms, scatter plots of ISI series, and ISI histograms of 3 STN neurons presented in A. (E and F) Overexpression of HCN2 in the STN decreased the CV of ISIs and the number of bursts and increased interburst intervals of STN neurons, whereas downregulation of HCN2 increased their CV of ISIs and the number of bursts and decreased interburst intervals (E, n = 30; F, n = 15). (G) Three continuous oscilloscope traces show the firings of an STN neuron in a PD rat before, during, and after high-frequency stimulation (125 Hz, 70 μA, 80 μs pulse width). Red arrowheads in the zoomed-in portion of the spike train during DBS indicate firing spikes of the recorded STN neuron. Insets represent 15 superimposed traces of spike waveforms for the unit, i.e., 5 traces before (green), 5 traces during (red), and 5 traces after (blue) stimulation. (H–J) Autocorrelation histograms, scatter plots of ISI series, and ISI histograms of the STN neuron presented in G. (K and L) DBS decreased the CV of ISIs (n = 25) and the number of bursts and increased interburst intervals of STN neurons (n = 15), indicating DBS regularizes the firing pattern of STN neurons. Data are represented as mean ± SEM. ***P < 0.001, 1-way ANOVA with Newman-Keuls post hoc test (E, F, K and L).

Figure 7. Downregulation of HCN2 attenuates STN-DBS–induced amelioration of motor dysfunction, firing patterns, and β oscillations in free-moving PD rats.

(A) Locomotor traces of PD rats in an open field. STN-DBS increased total movement distance of PD rats, whereas downregulation of HCN2 in STN abolished DBS-induced motor amelioration (n = 5). (B) STN-DBS increased histamine release in STN of PD rats in open field (n = 7). (C) Three continuous oscilloscope traces show firings of 3 STN neurons in free-moving PD rats treated with control virus, control virus plus STN-DBS, or downregulation of HCN2 virus plus STN-DBS. Red arrowheads indicate firing spikes of recorded STN neurons during DBS. Insets represent 5 superimposed traces of spike waveforms for each unit. (D–F) Autocorrelation histograms, scatter plots of ISI series, and ISI histograms of 3 STN neurons presented in C. (G and H) STN-DBS decreased CV of ISIs and number of bursts and increased interburst intervals of STN neurons in free-moving PD rats, whereas downregulation of HCN2 blocked STN-DBS–induced regularization of neuronal firing patterns (n = 15). (I) Power spectrograms of simultaneously recorded local field potentials in STN of these free-moving PD rats in the open field. (J) Power spectral distribution of local field potentials recorded from STN. Gray box indicates the classic β band (15–25 Hz). A clear increase of power in the β band was found in the PD rats. STN-DBS significantly alleviated dominant β band oscillatory activities in PD rats, whereas downregulation of HCN2 abolished STN-DBS–induced suppression of excessive β oscillations (n = 15). Data are represented as mean ± SEM. ***P < 0.001, 1-way ANOVA with Newman-Keuls post hoc test (A, G, H, and J) and 2-tailed paired t test (B).