Academic Editor: Graham Pawelec
Background: We obtained microelectrode recordings from four patients
with intractable aggressivity who underwent surgery at posteromedial hypothalamus under general anaesthesia. We
described two general types of extracellular action potentials (EAPs):
typical/canonical and atypical. Methods: We analysed 337 units and 67
traces, which were characterized by the mean action potential (mAP). For the
first phase, depolarization and repolarization, we computed amplitudes (V
Deep brain stimulation (DBS) has been demonstrated to be an effective surgical treatment for several movement disorders (e.g., Parkinson’s disease, essential tremor, and dystonia) and appears to be promising for other pathologies, such as epilepsy, pain, major depression, and Alzheimer’s disease. The process for all these diseases is to implant electrodes at different targets to modify the pathological activity in neural circuits by means of electrical stimulation. Microelectrode recording (MER) has long been known to be a reliable technique to identify neural structures .
One of these pathologies potentially treatable by DBS is intractable aggressivity, characterized by severe cases of unprovoked aggressive behaviour, usually associated with some degree of mental impairment and gross brain damage . The aetiology can be due to genetic origin, perinatal insults, brain malformation, or posttraumatic, postencephalitic or epileptic seizures and is usually accompanied by self-aggressiveness, hyperkinesia, and destruction of objects. Patients usually need to be institutionalized and managed with major restraint measures. An early successful surgical treatment was posteromedial hypothalamomy [2, 3]. Deep brain stimulation (DBS) of the posteromedial hypothalamus (PMH) has widely replaced ablative procedures because the clinical benefits appear to be similar, but the treatment can be titrated, is reversible, and has a low risk of complications [4, 5, 6, 7].
The human hypothalamus is a complex structure composed of a number of groups of nuclei . The identification of hypothalamic nuclei is particularly important to obtain good functional outcomes with DBS regarding optimizing battery life and decreasing secondary effects, especially considering that very sensitive nuclei related to arousal, circadian rhythms and hormone release are very close.
Most brain regions used for DBS have been described in terms of discharge patterns [9, 10, 11, 12, 13, 14, 15, 16], but less attention has been devoted to the analysis of extracellular action potentials (EAPs), with the exception of the pedunculopontine nucleus [17, 18] in a very limited way and some nuclei in the thalamus  and hypothalamus .
The hypothalamus is one of the least studied targets for DBS, probably because the pathologies treated with DBS in the hypothalamus have low prevalence. Descriptions of discharge patterns have been recently provided . However, it have been described, for the first time, features of EAPs from the posteromedial hypothalamus (PMH) and adjacent regions . In that work, it was described for the first time a type of cell with a highly anomalous EAP morphology, which was referred to as atypical (i.e., atypical mean action potential or amAP). Notably, amAPs were found mainly in the PMH, with lower concentrations in the regions above or below the PMH.
The morphology of amAPs is not the only electrophysiological feature of this region that is difficult to explain, as amAPs can be indicated by the presence of negative EAPs or the existence of a first phase that precedes the depolarization phase.
In this study, we aimed to analyse the electrophysiological features of amAPs recorded from the hypothalamus in anaesthetized patients. A detailed description of conventional mAPs will be used as a cornerstone for comparisons to amAPs.
Acronyms have been enlisted in Abbreviations.
We studied 4 patients undergoing DBS treatment at PMH for intractable aggressivity (see Table 1 for clinical information).
|Patient||Age (yrs)||Sex||Intellectual capacity||Medical history||Medication||MRI|
|1||22||F||severe mental retardation||cluster headache, epilepsy||TPM, CLZ, RIS, LVM, OLZ||moderate diffuse cortico-subcortical atrophy; pineal cyst|
|2||22||M||moderate mental retardation||perinatal hypoxia||GBP, VPA, CYP, Li, OLZ||normal|
|3||48||M||moderate mental retardation||OCD, AVM, complex partial seizure||CBM, GBP, ZPX, CTP, CLZ||extensive encephalomalacia in right temporal lobe|
|4||37||F||severe mental retardation||epilepsy||LVM, OLZ, TPM, RIS, ARP||normal|
|ARP, arpiprazole; AVM, arterio-venous malformation; CBM, carbamacepine; CLZ, clorazepate; CTP, citalopram; CYP, cyproterone; GBP, gabapentine; Li, lithium; LVM, levomepromazine; MRI, magnetic resonance imaging; OCD, obsessive-compulsive disorder; OLZ, olanzapine; RIS, risperidone; TPM, topiramate; VPA, valproic acid; ZPX, zuclopenthixol.|
All the patients were operated on while under general anesthesia using propofol
The hypothalamus was identified using a 1.5 T magnetic resonance imaging (MRI,
General Electric®, Fairfield, CT, USA), and the coordinates were
located stereotactically with a neuronavigator (BrainLab®,
Feldkirchen, Germany). The coordinates were calculated by fusing the MRI image
and computed tomography scan according to the Schaltenbrand-Wahren map . For
hypothalamic DBS electrode placement, a tentative initial target was selected in
the posterolateral hypothalamus according to the Sano’s triangle (x = 2, y = 0, z
= –2). All the coordinates (in mm) refer to the mid-intercommissural AC-PC line
(anterior commissure-posterior commissure). Neuronal recordings
(Leadpoint®, Minneapolis, MN, USA) were obtained beginning 10 mm
above the target and progressing in steps of 0.5 mm. MERs (FHC®,
Maine, USA) were obtained until 2 mm below the theoretical target. Impedance was
always above 900 k
MERs were obtained through 3–4 microelectrodes separated by 2 mm. A microdrive was fixed to a stereotactic Leksell Coordinate Frame (Elekta®, Stockholm, Sweden). The bandwidth for spontaneous activity was 200 Hz–5 kHz, with a sample rate of 24 kHz. The notch filter was off. PMH region was identified by MER and response to electrical stimulation [15, 23]. After the PMH was identified, a quadripolar DBS electrode was implanted, with a programmable stimulator placed in an abdominal or pectoral location.
The reconstruction of the trajectory was described in detail elsewhere . Anteroposterior and lateral coordinates were obtained from the post-op MRI performed one month after surgery. Using these points and the stereo-tactic angles, we reconstructed the real trajectory of the electrode in a three-dimensional space in 1-mm intervals.
Data were exported as ASCII files, and analyses were performed off-line. The
recordings spanned 30–90 s (72–216
The polarity of the potentials was defined as positive (P) upward and negative (N) downward and was identified by order of appearance.
(1) EAPs must have two phases (depolarization and repolarization); therefore, we
identified a tentative EAP when a positive/negative (P/N) phase was followed by a
negative/positive (N/P) phase in a period of 0.25–0.65 ms. EAPs were defined as
(2) For every EAP, we measured the maximum (Vmax) and minimum
voltages (Vmin, in
(3) Construction of the mean action potential (mAP) from all the EAPs from the
same cluster. A minimum of 10 EAPs was averaged. We identified hallmark points in
mAPs. Every phase can be characterized by its polarity (P/N), duration and
To compare the structure of different kind of mAP we have considered features of
the first phase, i.e., duration (d
We have built amplitude and duration ratios to characterize canonical forms of
mAP. So, we have
All analyses were performed in homemade MATLAB®R2018 (Natick, MA, USA) scripts.
Kurtosis (K) was computed for every group, and only values between 2 and 8 were acceptable for the homogeneous group . Extreme outliers were removed. Statistical analysis was applied only to these groups.
Statistical comparisons between groups were performed using the Mann-Whitney U
test or Kruskal-Wallis one-way ANOVA on ranks if normality failed. In the last
case, Dunn’s method was used for all pairwise post hoc comparisons. Normality was
evaluated using the Kolmogorov-Smirnov test. Chi-square test (
Pearson’s correlation coefficient was used to study linear dependence between
variables. Linear regression significance was evaluated by means a contrast
hypothesis against the null hypothesis
This describes a t-Student distribution with n-2 freedom degrees .
SigmaStat® 3.5 software (Point Richmond, CA, USA) and MATLAB were used for statistical analyses.
The significance level was set at p = 0.05, and the results are shown
as the mean
We analysed traces from 9 consecutive locations spanning 4.0 mm from 7
trajectories. Overall, we analysed 337 units and 67 raw traces. Given that each
mAP was composed of 34.8
Similar to thalamus recordings, in the hypothalamus, most mAPs were
characterized by the following features: (i) a higher amplitude positive/negative
phase followed by (ii) a phase of the opposite polarity, and (iii) the first
phase was shorter and included the dV
Correlations between pairs of variables. (A) V
We more deeply analysed the structure of the mAPs, defined as the arrangement of the components composing each of the waveforms . Excluding atypical mAPs (amAPs), most of the mAPs were positive, 260/295 (81.1%), and fewer were negative, 25/290 (19.9%). Atypical mAPs were recorded in 42/337 cases (12.5%). Most of these atypical waveforms were positive, but in other cases, the polarity was difficult to identify. Moreover, they presented structures clearly different from the other conventional types. Negative mAPs were recorded simultaneously with positive ones. Usually, the mAP had more than two phases. Only 120/337 (35.6%) mAPs showed 2 phases. On the other hand, a more frequent occurrence was to observe either a small positive or negative deflection before the main component, which resulted in a three-phase structure. In 113/337 (38.3%) cases, an N1P1N2 structure was observed, followed in 106/337 (35.9%) cases with a P1N1 structure, and a P1P2N1 structure in 41/337 (12.2%), an amAP in 42/337 (12.5%), an N1P1 structure in 14/337 (4.7%), an P1N1P2 structure in 12/337 (4.1%), and an N1N2P1 structure in 9/337 (3.1%) cases.
Theoretical considerations and empirical facts show that canonical forms of action potentials are composed of a high depolarization phase followed by a repolarization phase. Considering that both phases are part of the same process [28, 29, 30], a high correlation between them would be expected. The maximum and minimum values of the first derivative were located during the rising and falling periods of the depolarizing phase, respectively.
We plotted linear correlations between the different variables describing the P1N1 (n = 106), N1P1 (n = 14), N1P1N2 (n = 113), P1P2N1 (n = 41), N1N2P1 (9) and P1N1P2 (n = 12) cells in Fig. 1. We have added a new figure at Appendix (Appendix Fig. 7) to show the network for every kind of mAPs. Surprisingly, practically no correlations were observed between the durations of the phases or the durations and amplitudes (except for P1N1).
All the cells showed a strong relationship between V
We assessed these relationships as amplitude and duration ratios. We show these
ratios to identify constant relationships between cells. In Fig. 2, we compare
the amplitude and duration ratios for all typical cells. Considering all the
pairs between each kind of mAP and each ratio, we have
Amplitude and duration ratios for all the typical
mAPs. No differences were observed for ANOVA on ranks, with the exceptions of
Therefore, these ratios can be considered nearly constant for different kinds of
cells. It is quite interesting to observe that although no correlations between
With these results, we defined the properties of cells considered canonical as
those cells with (i) at least two opposite phases, with the higher, which is
always the first of the two, considered depolarization, followed by a
repolarizing phase; (ii)
Nevertheless, most of the recorded cells (74.4%) showed an initial phase prior to the depolarization phase. For all of these cells, the 5 conditions stated above were applicable, and therefore, these cells can be included as typical or canonical cells. We analysed the different correlations between pairs of magnitudes affecting the first phase (Fig. 3) and other properties of the APs. However, none of these correlations were significant.
Correlations between the FP variable and other
variables. (A) V
Therefore, there were no correlations between FP and variables characterizing
the depolarization or repolarization phases. Moreover, the coefficients of
These data suggest that the first phase was not associated with the same dynamic process of action potentials responsible for depolarization and repolarization.
As previously stated, up to 12.5% of cases were amAPs, a percentage that cannot
be considered anecdotal. Besides, this kind of cells was recorded from all the
patients. As with the canonical cells, we considered depolarization as the phase
Different phases of mAPs (red lines) and relationships with the first derivative (blue lines) for canonical and atypical cells. (A) P1N1 (B) N1P1N2 (C) P1P2N1 to (D) N1P1 cells for comparison with atypical cells in (E) wP1N1 (F) N1P1NLF (G) PbiN1 and (H) NbiP1. Horizontal dashed arrows indicate periods, and vertical dashed arrows indicate amplitudes of phases. Black dashed lines mark the maximum and minimum values of the first derivatives.
The observed amAPs have been named wide P1N1 (wP1N1, Fig. 4E) cells, cells with
a last phase (N1P1NLF, Fig. 4F), and positive bimodal (PbiN1, Fig. 4G) and
negative bimodal (NbiP1, Fig. 4H) cells. Each type of cell is plotted below its
canonical cell counterpart to clarify the difference. Explicitly, wP1N1 cells
(2/42, 5%) have a longer d
The first aspect we had to assess with respect to these amAPs was the possibility that such strange morphologies would arise from an erroneous sorting method that mixed different canonical forms. Therefore, it was extremely important to show atypical cells obtained from raw records. Fig. 5 shows three examples of amAPs obtained directly from the raw records before any sorting process.
Atypical extracellular action potentialsrecorded directly from the raw records before sorting. (A) NbiP1 (B) PbiN1 and (C) N1P1NLF. The horizontal lines under each record are expanded in the bottom panels. The horizontal calibration bar for every column corresponds from the upper to the lower row.
Therefore, it can be concluded that amAPs are truly extracellular action
potentials and were not artificially constructed. In this sense, the amplitude
and duration ratios of the amAPs were quite different from those of the canonical
mAPs . From a total of 14 ratios, in 10 cases, a significant difference was
observed (71%); a difference was observed with 6/7 amplitude ratios (86%), with
the only exception being V
The structures of atypical PbiN1, NbiP1 and wN1P1 cells suggested a composition of simple mAPs. In fact, a summation of two P1N1 cells delayed by 0.20–0.24 ms resulted in mAP quite similar to PbiN1 and wP1N1 (Fig. 6B,C). The same process using two consecutive N1P1 cells gave rise to a structure highly similar to NbiP1 (Fig. 6D).
Composition of atypical mAPs from simple cells. (A) Theoretical cell (not actually recorded) with a delay between unitary mAPs = 0.16 ms (B) wP1N1, delay = 0.21 ms (C) PbiN1, delay = 0.24 ms (D) NbiP1, delay = 0.24 ms. Blue = true unitary mAPs at different delays; red = theoretical mAPs formed by combining the two unitary mAPs; black = recorded amAP.
Unfortunately, we had no hypothesis about the sources leading to N1P1NLP cells, which are the most frequent amAPs.
To the best of our knowledge, this is the first study describing in detail the features of the atypical structure of EAPs in the human brain and, specifically, in the hypothalamus.
Recently, it has been shown that the types of mAPs in the hypothalamus are similar to those described in the thalamus [19, 20], with the majority of cells showing three phases. Despite the similar morphology in both brain structures, the properties of the mAPs were different, showing that mAP morphology is region specific. However, in the thalamus, we did not record the amAPs that were recorded in the hypothalamus . This fact adds another element of regional specificity regarding the structure of action potentials. EAPs can provide information about several properties of intracellularly recorded action potentials [28, 29]; therefore, the MER goal should not exclusively consider EAPs as binary events (present/absent). To date, scarce attention has been devoted to the analysis of EAPs. In fact, only EAP width has been reported from recordings in the pedunculopontine nucleus in humans, and a bimodal distribution has been observed with a longer APs attributed to cholinergic neurons and shorter APs attributed to glutamatergic transmission [18, 30, 31]. However, no other properties have been analysed (number of phases, features of phases, derivatives, etc.) until our previous studies in thalamic nuclei  and hypothalamic nuclei .
We have analysed in detail the structure of canonical forms of mAPs in the
hypothalamus, and we have identified five features defining conventional or
canonical mAPs. These properties can be well fitted to the described basis of
neuronal excitability in vivo by means of quantitative methods [32, 33, 34, 35]. The tight
relationship between V
The ratios of amplitudes and durations for the several phases of the canonical
forms have been proven to be a robust way to characterize typical mAPs. It is
quite interesting to observe that different types of mAPs share similar values of
The shape of the EAP is similar and proportional to the total transmembrane
current from the perisomatic region . However, as we previously described in
the thalamus  and hypothalamus , most cells have a small phase before
depolarization. This first phase can be observed with either positive or negative
polarities. Given that quantitative and experimental data have shown the presence
of capacitive current prior to the large depolarizing phase, this origin could be
hypothesized for the first phase described here. However, capacitive current is
elicited by current spreading to dendrites from the soma and is always of the
opposite polarity as depolarization; therefore, we would obtain only N1P1N2 and
P1N1P2 types. Thus, capacitive current cannot explain cells such as P1P2N1
(12.2%) and N1N2P1 (3.1%). Moreover, the magnitude of this current is between
10 and 50 times lower than the perisomatic current and cannot explain the
amplitude and duration of the FP described here. Another possibility could be
that the first phase is related to postsynaptic potentials, which can explain the
difference in polarity. This possibility is reinforced by the lack of correlation
and the greater variance for V
Another possibility to explain N1P1N2 cell morphology to capacitive or synaptic activity could be consider these kinds of cells as EAPs originating from axons, not the perisomatic region. Referential recordings from nonmyelinated fibres result in waveforms quite similar to a smaller first negative phase, followed by a higher positive and finally a third negative longer phase . If this kind of mAP would be, in fact, in axons, it could be expected that its percentage would be greater in regions richer in white fibres, such as the hypothalamus (i.e., caudate-pallidal fibres and lenticular, tuberomamillar and dorsal longitudinal fascicles go through the hypothalamus), and lower in nuclei mainly composed of dendrite-soma structures, such as the basal ganglia . This was true, because in the thalamus, the percentage of N1P1N2 was 19.4% (216/1114 cells), while in the hypothalamus, this percentage was nearly double (37.6%, 53/141 cells) . In fact, the initial axon segment is the dominant contributor to the extracellular action potentials of cultured neurons .
As previously described in the thalamus, negative mAPs have also been described in the hypothalamus. Absolute values of amplitude and duration for repolarization (what we to consider as the negative phase) are similar to repolarization in positive cells. If depolarization originates from the transmembrane current going into the cell [35, 38], we should think that negative depolarization originates from the transmembrane current coming from inside the cell. However, this case has not been described to date. The presence of these negative cells is high enough to discard an anecdotal finding; therefore, the transmembrane sources of current for these cells remain to be explained. In animal cortical recordings, a high amplitude positive action potential different from conventional negative spikes has been described, and the features of both types of spikes, such as amplitude and duration, were clearly different . However, these findings were not observed in our case, where the properties of the positive and negative mAPs (absolute magnitudes for amplitudes and phase durations) were on the same order of magnitude.
To the best of our knowledge, this is the first description of EAPs that
significantly differs from that conventionally described in animals and humans or
quantitatively predicted [32, 34, 40]. First, it is important to keep in mind
that despite their anomalous structure, they are real action potentials and not
artefacts from erroneous sorting. Although we have denominated the amAPs in a
similar way to canonical forms, the amplitudes and durations are strikingly
different. The amAPs PbiN1 and NbiP1 have three well-defined phases. However, the
first phase cannot be considered a canonical first phase because it is usually
the higher amplitude phase. Obviously, neither a postsynaptic potential nor
capacitive current can be so high. In fact, both dV
Another possibility to explain the broad mAP could be the participation of calcium currents. It is well known that this type of conductance enlarges the duration of action potentials and can be located at dendritic sites .
The most frequently found amAP was N1P1N2LP. Although slightly similar at first
sight to N1P1N2 canonical cells, there were some differences. For the canonical
mAP, the ratio V
Nevertheless, it is important to understand that our data were obtained from a small number of patients. Although these data seem to be robust, a larger cohort study is needed to corroborate our interpretation of various EAP patterns as atypical and to confirm a connection between the presence of atypical potentials and the target identification. To further corroborate our interpretations of the EAP, simulations with neural models incorporating ion channel dynamics could be helpful.
EAPs of hypothalamic cells in anaesthetized humans can be divided into canonical
forms, sharing a set of properties that are highly robust, mainly concerning the
relationship between V
The MATLAB® script is available upon request from corresponding author.
amAP, Atypical Mean Action Potential; AP, Action Potential; DBS, Deep Brain
JP is responsible of the idea. LV-Z and EM-A participated in data collection. JP developed the analytical methods and EM-A and LV-Z participated also in analysis and interpretation, and JP was responsible for manuscript preparation. All authors have approved the submitted version of this manuscript.
The experimental procedure was approved by the medical ethics review board of the Hospital Universitario de La Princesa and was deemed “care as usual”. Under these circumstances, written informed consent was not required because all procedures were done by clinical necessity and analysis was off-line. The study was accomplished according to standards for human research and in accordance with the Helsinki Declaration.
Authors want to acknowledge the collaboration during surgeries of neurosurgeons Marta Navas and Cristina Torres and anaesthesiologists María Luisa Meilán and Eva de Dios and Angel Nuñez by its comments during the elaboration of the manuscript.
This research received no external funding.
The authors declare no conflict of interest. JP and LV-Z are serving as the guest editor of this journal. We declare that JP and LV-Z had no involvement in the peer review of this article and has no access to information regarding its peer review. Full responsibility for the editorial process for this article was delegated to GP.
To assess the influence of the signal-to-noise ratio (SNR) in the relations
between different parts of mAP we have mixed different degrees of known noise
We have simulated a P1N1 mAP fitting the next properties (i) two opposite phases
of depolarization followed by repolarization, (ii)
To simulate noise, we have chosen a function like this
We have plotted these results at Appendix Fig. 8. We can observe that there was
a very good correlation between A and V
But, not only no correlation between durations were obtained, besides the
coefficient of variation was higher for d
Network plot for correlations between variables for all the kind of mAP. The strength of correlation is indicated by the thickness of line.
Different mixtures of the model of mAP with
noise. (A) Signal without noise. (B) SNR = 17.1, A = 5.2
Relationships between variables from different
mixtures of mAP + noise. (A) A vs V