Spreading depolarization triggered by elevated potassium is weak or absent in the rodent lower brain

Neocortical slice preparation

Male C57 Black mice or Sprague-Dawley rats, 21–50 days old (Charles River, St. Constant, Quebec) were cared for in accordance with the principles and guidelines of the Canadian Council on Animal Care as overseen by the Queen’s University Animal Care Committee. This study complied with the ARRIVE guidelines (Animal Research: Reporting in Vivo Experiments). Rodents were housed in a controlled environment (22 ± 1℃, 12 h light:12 h dark cycle) with food (Purina rat chow) and water supplied ad libitum. Rats were placed in a rodent restrainer (DecapiCone; Braintree Scientific, Inc.) and guillotined. The brain was excised and placed in ice-cold oxygenated (95% O₂–5% CO₂) artificial cerebrospinal fluid (aCSF) with NaCl replaced by sucrose. Coronal slices (400 µm) were taken using a vibrating blade microtome (Leica VT1000S). Five to seven slices were transferred to a submerged net in a beaker containing regular aCSF gassed with O₂–CO₂. The slices were then slowly warmed over 1 h to 32℃ prior to experimentation at 33–34℃.

Experimental solutions and drugs

The aCSF contained (in mM) 120 NaCl, 3.3 KCl, 26 NaHCO₃, 1.3 MgSO₄ 7H₂O, 1.2 NaH₂PO₄, 1.8 CaCl₂, and 11D-glucose. All were dissolved in double-distilled water at pH 7.3–7.4. The aCSF was used for incubation and as a vehicle to administer experimental solutions. For high-K⁺ aCSF, 9.6, 26, or 52 mM KCl replaced equimolar NaCl. Kynurenic acid (2 mM) was added to control aCSF or to elevated-KCl ACSF as required. All drugs were from the Sigma-Aldrich. During an experiment, a slice were submerged in oxygenated aCSF flowing at a rate of 3–4 ml/min at 33–34℃.

Imaging intrinsic optical signals

Changes in light transmittance (LT) through live brain slices are generated as LT is increased by brain cell swelling or reduced by dendritic beading which increases light scatter.^32,33 Using previously described techniques,^6,23 ΔLT was monitored and recorded. Graphical and statistical analyses of data were carried out using Excel. Images were imported and figures were prepared using CorelDraw. A ΔLT value from each brain region of interest (ROI) is an average of that region (∼500 µm²). The data from the imaging experiments were analyzed such that changes in LT of a given ROI were expressed as percent changes of the T_cont for that region, taken from the control image. This normalized the graphical data across the different regions of the neocortex, which was necessary because of the variation in opacity that causes different initial LT values (T_cont). The means were calculated and reported using a paired Student’s t-test for statistical analysis.

Electrophysiology

Micropipettes were pulled from borosilicate glass (OD 1.2 mm, ID 0.68 mm; World Precision Instruments) to a resistance of 3–6 MΩ. The internal pipette solution contained (in mM) 125 K-gluconate, 10 KCl, 2 MgCl₂, 5.5 EGTA, 10 HEPES, 2 Na-ATP, and 0.1 CaCl₂ (pH was adjusted to 7.3 with KOH). A 14 mV junction potential was corrected prior to achieving whole-cell configuration. Whole-cell patched neurons were analyzed if they displayed stable resting membrane potentials and if the series resistance could be sufficiently compensated. Recordings were from neurons visualized with near-infrared illumination and Dodt gradient contrast optics (Luigs and Neumann, Ratingen, Germany) through an upright microscope (Axoscope 2FS, Zeiss) with a × 40 immersion objective lens. Neurons were recorded from neocortical layers 2–3, striatum, thalamus, the hypothalamus (supraoptic nucleus, paraventricular nucleus, suprachiasmatic nucleus), the mesencephalic nucleus (MES) of midbrain-pons, locus coeruleus (LC) of midbrain-pons, dorsal nucleus solitarius (SolN) of medulla oblongata, and dorsal motor nucleus (DMV) of medulla oblongata. All recordings were acquired in current clamp mode of an Axoclamp 2 A amplifier and a Digidata 1322 A/D converter. Clampex 10 software was used for data acquisition with subsequent analysis using Clampfit 10 software. Sampling frequency was 10 kHz and low pass filtering was with an external Bessel filter (LPF 202 a) at 2 kHz (Molecular Devices). Tabulated data of the electrophysiological properties of higher and lower neurons exposed to OGD can be found at http://hdl.handle.net/1974/7578. A subset of these neurons for this study were first exposed to elevated [K⁺]_o, and their properties are listed in Table 1. All of these neurons returned to their baseline properties following the brief K⁺ exposure. Electrophysiological data were presented as means ± standard deviation.

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Table 1.

Whole-cell parameters during 2–3 min of [K⁺]_o elevated to 52 mM recorded from “higher” pyramidal neurons (PYR) in neocortex and CA1 region as well as from spiny neurons in striatum (total n = 14).

Neuron type	n	Rmp (mV)	AP ampl (mV)	Rin (MΩ)	Depol rate (mV/min)	SDKt rate (mV/s)
Neocortex PYR	5	Mean −82	78	82	NA	10 to 25
		SD ± 2.7	7.9	12.1
CA1 PYR	5	mean −73	82	97	NA	3 to 22
		SD ± 2.5	13.1	17.8
Striatum	4	mean −71	80	54	NA	12 to 30
		SD ± 4	10.5	15.2
Hypothal MNC	1	−62	94	569	15	NA
Hypothal Parvo	1	−51	70	778	18	NA
Hypothal SCN	1	−50	80	676	15	NA
Midbrain MES	1	−54	77	52	20	NA
Midbrain LC	1	−48	89	181	20	NA
Medulla SolN	1	−47	94	720	17.5	NA
Medulla DVM	1	−48	98	284	17.5	NA

Imaging spreading depression

To generally assess the regional propensity of the CNS to undergo spreading depression, we exposed live coronal brain slices to elevated [K⁺]_o while imaging changes in light transmittance (ΔLT). Elevating bath [K⁺]_o to 26 mM for 3 to 5 min evokes a front of spreading depression (SD_Kt) in the mouse brain slice (n = 14 mice). The increased LT is caused by the swelling of neurons as the cells depolarize with astrocytes swelling secondarily. ³⁴ The SD_Kt front (Figure 1(a), arrows) propagates in neocortex (NC) and hippocampus (Hp) but much less so in the brainstem with the exception of dorsal brainstem gray specifically, the superficial superior colliculus (Figure 1(a), SC), superficial inferior colliculi (IC) and periaqueductal gray (Figure 1(a), PAG), solitary nucleus (SolN), and tegmental nucleus (TN). All regions recover from SD_Kt-associated swelling. OPEN IN VIEWER

SD_Kt was induced by exposing coronal mouse brain slices to 26 mM K⁺ aCSF for 3–5 min. Figure 1(b) and 1(c) illustrates several regional ΔLT time courses during simultaneous SD_Kt events. The CA1 region (46.5 ± 10.1%) and cerebellar cortex (54.0 ± 20.8%) had a significantly higher mean LT peak compared with other regions (*p < 0.05) (Figure 2(a)). The mean LT peak in SC was 34.9 ± 11.3% which was similar to the neocortex (33.9 ± 14.4%). PAG had a mean LT peak of 29.3 ± 17.6%. In addition, there was little difference in the mean LT peak among IC (32.4 ± 11.3%), TN (30.1 ± 7.3%), and SolN (32.0 ± 13.0%). OPEN IN VIEWER

There was no significant difference in SD_Kt onset time among the responding regions (Figure 2(b)). The SD_Kt propagation rate in brainstem nuclei (Figure 2(c)) was 1.9 ± 1.2 mm/min in SC, 1.4 ± 1.5 mm/min in IC, 0.7 ± 0.2 mm/min in PAG, and 0.5 ± 0.2 mm/min in TN. These rates were significantly slower than the higher brain regions of neocortex (4.1 ± 0.8 mm/min), CA1 (3.4 ± 2.0 mm/min), and cerebellar cortex (2.6 ± 2.3 mm/min). SD_Kt spread through SC and IC at similar speeds. PAG, TN, and IC displayed significantly slower SD_Kt compared with neocortex (p < 0.05) (Figure 2(c)). Comparatively, the propagation rate was neocortex > CA1 > cerebellar cortex > SC > IC > PAG > TN. The small size of the solitary nuclei made it difficult to measure a rate.

Kynurenic acid did not significantly affect the mean peak LT in neocortex (n = 4), CA1 (n = 3), SC (n = 7), cerebellar cortex (n = 6), or IC (n = 4) (number of mice = 7) (Figure 2(a)). SD_Kt onset in the cerebellar cortex and neocortex were delayed by kynurenate compared with control (*p < 0.05). Kynurenic acid did not alter SD_Kt onset in CA1, SC, or IC (Figure 2(b)). In addition, the SD_Kt propagation rate tended to decrease in most regions including neocortex, CA1, SC, cerebellar cortex, and IC but only the propagation in neocortex and the CA1 region was significantly different (*p < 0.001) (Figure 2(c)). In some cerebellar and IC slices, we detected increases in LT but without obvious SD_Kt propagation (not shown). No SD_Kt was observed after kynurenate pre-treatment in PAG (6/6 slices) and SolN (5/5 slices). Only 1/9 TN slices displayed a signal with kynurenic acid treatment. The distinct LT increases in brainstem nuclei that emulated higher gray matter were only observed near the dorsal surface of brainstem and not in hypothalamus.

Intracellular recordings

As described in numerous previous studies, pyramidal neurons in layers II–III of the neocortex depolarize in response to elevated [K⁺]_o, leading to spreading depression (SD_Kt) that approaches zero millivolts (Figure 3(a)). In response to an initial 9.6 mM K⁺ exposure, a pyramidal cell passively depolarizes slightly and repolarizes in control aCSF. Exposure to 26 mM K⁺ elicits more depolarization with firing and then spike inactivation, before a plateau of −48 mV is reached. SD_Kt onset is aborted just as control aCSF reaches the slice (arrow). A second exposure again evokes firing and spike inactivation, reaching a plateau at −48 mV. A steep secondary depolarization signals SD_Kt onset. The membrane potential again returns to resting level after aCSF return. In contrast, hypothalamic neurons slowly depolarize without the active SD_Kt event (Figure 3(b1)). A magnocellular neuron in the supraoptic nucleus (SON) depolarizes in 52 mM K⁺ aCSF. No SD_Kt event that approaches zero millivolts is evoked. After each exposure, the cell recovers. In Figure 3(b2), a parvocellular neuron in paraventricular nucleus (PVN) depolarizes in 52 mM [K⁺]_o aCSF. Action potentials inactivate while slowly reaching a plateau but no SD_Kt is rises. With return to control aCSF, it repolarizes. Similarly, a suprachiasmatic nucleus (SCN) neuron responds to 3 min of 52 mM [K⁺]_o and depolarizes to a plateau as action potentials inactivate (Figure 3(b3)), but does not generate SDKt. OPEN IN VIEWER

Exposing a neostriatal neuron to 100 s of 52 mM [K⁺]_o results in a rapid depolarization to near zero millivolts (Figure 4(a1)). A brief plateau at −30 mV (arrow) occurs before a second more rapid depolarization to near zero millivolts, typical of SD_Kt. Upon return of control aCSF, the membrane potential recovers. This contrasts to the typical response to oxygen/glucose deprivation by striatal neurons (Figure 4(a2)), where a typical “higher” neuron response is a rapid, complete, and irreversible “anoxic” depolarization (AD). In midbrain slices, a 3-min exposure to 52 mM [K⁺]_o elicited depolarization of a mesencephalic neuron (MES, n = 3) to −19 mV with loss of action potential firing (Figure 4(b1)). No SD_Kt is generated unlike in Figure 4(a1). Upon return of control aCSF, MES neurons return to their original resting membrane potential. Similarly in a locus ceruleus neuron, 3 min of 52 mM [K⁺]_o elicits depolarization and spike inactivation to −11 mV but no SD_Kt (Figure 4(b2)). Upon return of control aCSF, the LC neuron repolarizes. OPEN IN VIEWER

Figure 5 further contrasts a higher brain region (thalamus) with brainstem recordings from slices of medulla. A thalamic neuron responds to 120 s of 52 mM [K⁺]_o with a quick depolarization to near zero millivolts (Figure 5(a1)), typical of SD_Kt in higher neurons. The membrane potential returns to baseline upon return of control aCSF (not shown). As in Figure 4(a2), thalamic neurons also rapidly undergo AD when exposed to OGD (Figure 5(a2)). In lower neurons, a 3-min exposure to 52 mM [K⁺]_o elicits slow depolarization of a dNTS neuron with loss of action potential firing, before reaching a plateau of −30 mV (Figure 5(b1)). No SD_Kt is generated. With return of control aCSF, the SolN neuron repolarizes. Similarly, DVM neurons depolarize during 3 min of 52 mM [K⁺]_o, reaching a plateau of −29 ± 2.1 mV as firing stops (n = 8). Upon return of control aCSF, all DMV neurons repolarized to their original resting levels. The DMV neuron in Figure 5(b2) was subjected to two separate 52 mM [K⁺]_o exposures (3 min) that reached respective plateaus of −32 and −28 mV. As with the hypothalamic and brainstem neurons recorded in this study, no SD_Kt was generated (Table 1). The rate of depolarization from baseline to plateau by seven lower neurons during 2 min of 52 mM KCl exposure was 17.5 ± 2.0 mV/min. That is exceeding slow compared with the SD_Kt rate generated by higher neurons, which, once underway, ranges between 3 and 30 mV/s, as shown in Table 1. OPEN IN VIEWER

The role of swelling in SD

Both astrocytes and neurons swell during AD^33,41 and SD_Kt. ³⁴ Astrocytic swelling is more easily explained because their surface is studded with water channels that mediate passive water flux across their plasma membranes. ⁴² As [K⁺]_o rises during SD, K⁺ and Cl⁻ are taken up followed by osmotic water. But with CNS neurons, swelling is more complicated because their plasma membrane lacks functioning aquaporins and so they do not passively swell or shrink over many minutes^33,41 across a range of osmolalities (−80 to +80 milliosmoles). Osmotic stress can acutely arise over a short period of time in a number of clinical situations.^43,44 The accumulation of water within neurons under metabolic stress occurs in at least two general ways. First, many ion and amino acid co-transporters, most energetically linked to the Na/K pump, move water across plasma membrane as they carry out their normal functions,^45–47 even against its concentration gradient. ⁴⁸ Second, water is a catabolic by-product of metabolism and if the water pumping noted above fails as ischemia develops, the lack of neuronal aquaporins means accumulating water is trapped intracellularly. The result during metabolic stress is neuronal swelling because water cotransporters are disrupted so residual water produced would remain in the neuron.

Why does SD show a regional susceptibility?

The question arises as to why SD in higher gray matter is stronger and less reversible than in the lower gray matter. There is no obvious architectural reason why higher and lower regions differ in susceptibility to SD. Also there are no data indicating that higher brain regions consume more energy per unit volume than lower regions. Nor is there any evidence that a cortical neuron requires more energy than a brainstem neuron nor that neurons from higher vs. lower regions systematically differ in their ion channel composition. We think it more likely that there is a regional difference involving the molecular biology of Na⁺/K⁺ pump failure and the ensuing depolarization that results. The physiological cause of SD is pump failure or alternately, overriding the pump by raising [K⁺]_o. One approach is to look for regional variations in the expression of molecular transporters known to play important roles in maintaining normal metabolism.

For example, in situ hybridization data mined from the Allen Mouse Brain Atlas shows the regional expression pattern of the ATP1α1 pump and the glutamate transporter SLC17a7 (Figure 6). Gene expression primarily in higher brain regions is high for these two transporters or their related proteins, suggesting a potential specific interaction between them. Rose et al. ⁴⁹ proposed that the Na⁺/K⁺ pump interacts through its γ-subunit because inhibiting the ATPase also partly inhibits the glutamate transporter. Such an interaction could promote extracellular glutamate accumulation during Na/K pump failure, extending ischemic injury in higher brain regions. Therefore, regional variation in acute ischemic injury may not simply involve an up- or down-regulation of metabolic pumps but also how they interact. In comparison to a wide variety of ligand and voltage-gated channels that have been proposed to malfunction in migraine and stroke (yielding little mechanistic insight), transporters have only been minimally studied. OPEN IN VIEWER

In summary, SD may contribute to survival both at behavioral and neuronal levels and so be selected during vertebrate evolution for the same reason as in insects. Both the locust^25,26 and fruit-fly ²⁷ use SD to temporarily shut down their nervous system when temperature is too high or too low or when anoxia sets in. Similarly, we propose that SD arising from TBI elicits electrical silence and vasoconstriction in higher gray matter to preserve metabolic energy, to avoid epileptiform activity and to reduce hemorrhage. From a behavioral standpoint, a sudden loss of consciousness following TBI is preferable to seizure or uncoordinated movement in terms of avoiding the attention of enemies or predators. As a milder version of SD, we propose that SD_Kt is a vestigial response to local metabolic stress, and so it is generated in TBI-prone brain regions but is rare in hypothalamus and ventral brainstem.