Gap junction blockers attenuate beta oscillations and improve forelimb function in hemiparkinsonian rats.

Parkinson's disease (PD) is a neurodegenerative disease characterized by akinesia, bradykinesia, resting tremors and postural instability. Although various models have been developed to explain basal ganglia (BG) pathophysiology in PD, the recent reports that dominant beta (ß) oscillations (12-30Hz) in BG nuclei of PD patients and parkinsonian animals coincide with motor dysfunction has led to an emerging idea that these oscillations may be a characteristic of PD. Due to the recent realization of these oscillations, the cellular and network mechanism(s) that underlie this process remain ill-defined. Here, we postulate that gap junctions (GJs) can contribute to ß oscillations in the BG of hemiparkinsonian rats and inhibiting their activity will disrupt neuronal synchrony, diminish these oscillations and improve motor function. To test this, we injected the GJ blockers carbenoxolone (CBX) or octanol in the right globus pallidus externa (GPe) of anesthetized hemiparkinsonian rats and noted whether subsequent changes in ß oscillatory activity occurred using in vivo electrophysiology. We found that systemic treatment of 200mg/kg CBX attenuated normalized GPe ß oscillatory activity from 6.10±1.29 arbitrary units (A.U.) (pre-CBX) to 2.48±0.87 A.U. (post-CBX) with maximal attenuation occurring 90.0±20.5min after injection. The systemic treatment of octanol (350mg/kg) also decreased ß oscillatory activity in a similar manner to CBX treatment with ß oscillatory activity decreasing from 3.58±0.89 (pre-octanol) to 2.57±1.08 after octanol injection. Next, 1µl CBX (200mg/kg) was directly injected into the GPe of anesthetized hemiparkinsonian rats; 59.2±19.0min after injection, ß oscillations in this BG nucleus decreased from 3.62±1.17 A.U. to 1.67±0.62 A.U. Interestingly, we were able to elicit ß oscillations in the GPe of naive non-parkinsonian rats by increasing GJ activity with 1µl trimethylamine (TMA, 500nM). Finally, we systemically injected CBX (200mg/kg) into hemiparkinsonian rats which attenuated dominant ß oscillations in the right GPe and also improved left forepaw akinesia in the step test. Conversely, direct injection of TMA into the right GPe of naive rats induced contralateral left forelimb akinesia. Overall, our results suggest that GJs contribute to ß oscillations in the GPe of hemiparkinsonian rats.

Elevated potassium provides an ionic mechanism for deep brain stimulation in the hemiparkinsonian rat.

The mechanism of high-frequency stimulation used in deep brain stimulation (DBS) for Parkinson's disease (PD) has not been completely elucidated. Previously, high-frequency stimulation of the rat entopeduncular nucleus, a basal ganglia output nucleus, elicited an increase in [K(+)](e) to 18 mm, in vitro. In this study, we assessed whether elevated K(+) can elicit DBS-like therapeutic effects in hemiparkinsonian rats by employing the limb-use asymmetry test and the self-adjusting stepping test. We then identified how these effects were meditated with in-vivo and in-vitro electrophysiology. Forelimb akinesia improved in hemiparkinsonian rats undergoing both tests after 20 mm KCl injection into the substantia nigra pars reticulata (SNr) or the subthalamic nucleus. In the SNr, neuronal spiking activity decreased from 38.2 ± 1.2 to 14.6 ± 1.6 Hz and attenuated SNr beta-frequency (12-30 Hz) oscillations after K(+) treatment. These oscillations are commonly associated with akinesia/bradykinesia in patients with PD and animal models of PD. Pressure ejection of 20 mm KCl onto SNr neurons in vitro caused a depolarisation block and sustained quiescence of SNr activity. In conclusion, our data showed that elevated K(+) injection into the hemiparkinsonian rat SNr improved forelimb akinesia, which coincided with a decrease in SNr neuronal spiking activity and desynchronised activity in SNr beta frequency, and subsequently an overall increase in ventral medial thalamic neuronal activity. Moreover, these findings also suggest that elevated K(+) may provide an ionic mechanism that can contribute to the therapeutic effects of DBS for the motor treatment of advanced PD.

Deep brain stimulation of the substantia nigra pars reticulata improves forelimb akinesia in the hemiparkinsonian rat.

Deep brain stimulation (DBS) employing high-frequency stimulation (HFS) is commonly used in the globus pallidus interna (GPi) and the subthalamic nucleus (STN) for treating motor symptoms of patients with Parkinson's disease (PD). Although DBS improves motor function in most PD patients, disease progression and stimulation-induced nonmotor complications limit DBS in these areas. In this study, we assessed whether stimulation of the substantia nigra pars reticulata (SNr) improved motor function. Hemiparkinsonian rats predominantly touched with their unimpaired forepaw >90% of the time in the stepping and limb-use asymmetry tests. After SNr-HFS (150 Hz), rats touched equally with both forepaws, similar to naive and sham-lesioned rats. In vivo, SNr-HFS decreased beta oscillations (12-30 Hz) in the SNr of freely moving hemiparkinsonian rats and decreased SNr neuronal spiking activity from 28 ± 1.9 Hz before stimulation to 0.8 ± 1.9 Hz during DBS in anesthetized animals; also, neuronal spiking activity increased from 7 ± 1.6 to 18 ± 1.6 Hz in the ventromedial portion of the thalamus (VM), the primary SNr efferent. In addition, HFS of the SNr in brain slices from normal and reserpine-treated rat pups resulted in a depolarization block of SNr neuronal activity. We demonstrate improvement of forelimb akinesia with SNr-HFS and suggest that this motor effect may have resulted from the attenuation of SNr neuronal activity, decreased SNr beta oscillations, and increased activity of VM thalamic neurons, suggesting that the SNr may be a plausible DBS target for treating motor symptoms of DBS.

Deconstructing the neural and ionic involvement of seizure-like events in the striatal network.

Seizures occur in the basal ganglia (BG) of epileptic patients and in animal models of epilepsy, but there is relatively little known about how these events are gated and/or propagated through this structure. Here, we present and characterize a model of in vitro seizure-like events (SLEs) in the striatum by applying chemostimulants to brain slices from young rat pups. We found that bath perfusion of artificial cerebral spinal fluid (aCSF) containing 0.25 mM MgCl(2), 5mM KCl and 100 µM 4-aminopyridine (LM/HK/4AP) elicited recurrent hyper-excitability in striatal medium spiny neurons (MSNs) in the form of paroxysmal depolarization shifts (PDSs) with an amplitude of 27.8 ± 2.1 mV and a duration of 29.4 ± 3.7s. PDSs coincided with SLEs in the striatal network with an amplitude of 106.5 ± 11.3 µV, duration of 23.6 ± 3.2 s, and a spiking frequency of 7.9 ± 1.3 Hz. Notably, chemostimulant-induced MSN PDSs were predominantly observed at earlier ages (P7-11), whereas occurrence of MSN PDSs declined to 50% by P12 and were no longer noted after P14; antagonism of the cannabinoid receptor (CB1) with 10 µM LY 320135 along with perfusion of LM/HK/4AP in older animals (P14-15) was unable to elicit MSN PDSs and SLEs. PDSs and SLEs were blocked with 60 µM 2-amino-5-phosphonopentanoate (APV), an N-methyl-d-aspartate receptor (NMDAR) blocker, or with traditional anticonvulsants such as 100 µM phenytoin or 50 µM carbamazepine. Conversely, blockade of 2-amino-3-(5-methyl-3-oxo-1,2-oxazol-4-yl) propanoic acid receptors (AMPARs) with 10 µM CNQX or T- and L-type Ca(2+) channels with 50 µM NiCl(2) or 50 µM nimodipine, respectively, did not significantly change MSN PDS and SLE amplitudes, durations and frequencies seen with LM/HK/4AP treatment alone. Striatal SLEs were driven by MSN hyper-excitability and synchrony since neither the presence of 1µM scopolamine, a muscarinic acetylcholine (ACh) receptor inhibitor, nor selective inhibition of fast-spiking interneurons (FSIs) with 50µM IEM1460 had any significant effect on MSN PDSs and SLEs. Next, we physically isolated the striatum from cortical and thalamic input and found that the striatum was intrinsically capable of manifesting NMDAR-dependent SLEs. Altogether, the present study is the first to deconstruct how SLEs can form in the striatum by examining how MSN activity coincides with SLEs. It also highlights a previously unrecognized potential for the striatum to manifest SLEs in vitro, without involving the cortex and thalamus. From these findings, further hypotheses can be developed for studying the BG's role in seizure generation and propagation, which may lead to novel pharmacological targets for the treatment of epilepsy.

Isovaline, a rare amino acid, has anticonvulsant properties in two in vitro hippocampal seizure models by increasing interneuronal activity.

PURPOSE: We investigated whether RS-isovaline, a unique amino acid found in carbonaceous meteorites and presumed extraterrestrial, has anticonvulsant properties in rat hippocampal slices in vitro. METHODS: Extracellular recordings were obtained in the rat hippocampal CA1 pyramidal layer in two in vitro seizure models: perfusion of low (0.25 mm) Mg(2+) and high (5 mm) K(+) (LM/HK), or 100 µm 4-aminopyridine (4-AP). To investigate the underlying mechanisms of isovaline action, whole-cell recordings were obtained from CA1 pyramidal neurons and stratum oriens interneurons during 4-AP blockade of K(+) channels. KEY FINDINGS: Perfusion of LM/HK produced seizure-like events (SLEs) or stimulus-evoked primary afterdischarges (PADs) with amplitudes of 0.9 ± 0.1 mV lasting 80 ± 14 s. Application of isovaline (250 µm) for 20-30 min abolished SLEs and PADs or attenuated seizure amplitude and duration by 57.0 ± 9.0% and 57.0 ± 12.0%, respectively. Similar effects were seen with isovaline in the 4-AP seizure model. Isovaline alone increased interneuronal spontaneous spiking from 0.9 ± 0.3 to 3.2 ± 0.9 Hz, increased input resistance by 21.6 ± 8.1%, and depolarized the resting membrane potential by 8.0 ± 1.5 mV; no changes in the firing or electrical properties of pyramidal neurons were observed. Coapplication of 4-AP and isovaline increased interneuronal spontaneous spiking from 1.0 ± 0.6 to 2.6 ± 0.8 Hz, whereas pyramidal neuronal spiking activity decreased from 0.6 ± 0.4 to 0.2 ± 0.1 Hz. SIGNIFICANCE: Isovaline exhibited anticonvulsant properties in two hippocampal seizure models. This may lead to the development of a new class of anticonvulsants based on an unusual mechanism of action of this presumed extraterrestrial amino acid.

Elevated potassium elicits recurrent surges of large GABAA-receptor-mediated post-synaptic currents in hippocampal CA3 pyramidal neurons.

Previously, we found that rat hippocampal CA3 interneurons become hyperactive with increasing concentrations of extracellular K(+) up to 10 mM. However, it is unclear how this enhanced interneuronal activity affects pyramidal neurons. Here we voltage-clamped rat hippocampal CA3 pyramidal neurons in vitro at 0 mV to isolate γ-aminobutyric acid (GABA)-activated inhibitory post-synaptic currents (IPSCs) and measured these in artificial cerebrospinal fluid (aCSF) and with 10 mM K(+) bath perfusion. In aCSF, small IPSCs were present with amplitudes of 0.053 ± 0.007 nA and a frequency of 0.27 ± 0.14 Hz. With 10 mM K(+) perfusion, IPSCs increased greatly in frequency and amplitude, culminating in surge events with peak amplitudes of 0.56 ± 0.08 nA, that appeared and disappeared cyclically with durations lasting 2.02 ± 0.37 min repeatedly, up to 10 times over a 30-min bath perfusion of elevated K(+). These large IPSCs were GABA(A)-receptor mediated and did not involve significant desensitization of this receptor. Perfusion of a GABA transporter inhibitor (NO-711), glutamate receptor inhibitors CNQX and APV, or a gap junctional blocker (carbenoxolone) prevented the resurgence of large IPSCs. Pressure ejected sucrose resulted in the abolishment of subsequent surges. No elevated K(+)-mediated surges were observed in CA3 interneurons from the stratum oriens layer. In conclusion, these cyclic large IPSC events observable in CA3 pyramidal neurons in 10 mM KCl may be due to transient GABA depletion from continuously active interneuronal afferents.