*5.1. vmPFC Stimulation*

Hamani et al. published the first preclinical study of vmPFC-DBS in rats in 2010. They used the forced swim test (FST), which models "helplessness" in animals including anxiolytic-like and anti-anhedonic-like behavior. They found DBS reduced the immobility score in FST, indicating antidepressant-like e ffects. The authors attributed the behavioral changes to serotonergic function in the dorsal raphe nucleus (DRN) as lesions in this structure abolished the behavioral e ffects

in FST [91]. Another animal study in 2012 found the optimal stimulation frequency and amplitude of vmPFC-DBS was 130 Hz and 200 μA that produced anti-anhedonic-like e ffects and produced a charge density similar to DBS in humans [93]. They found a lesion in the DRN abolished the higher sucrose consumption due to DBS, even with a normal hippocampal brain-derived neurotrophic factor (BDNF) profile. They postulated that an interaction between BDNF and neurochemical substances potentiated the antidepressant-like response [92]. The anti-anhedonic-like e ffects of DBS were also supported in studies by Rea et al. and Edemann-Callesen et al. They conducted an intracranial, self-stimulation paradigm in Flinders sensitive line and Flinders resistant line rats to assess reward-seeking behaviors, which demonstrated that the anti-anhedonic-like e ffect of vmPFC-DBS was independent of the dopaminergic reward system [94,95]. Bruchim-Samuel et al. reported that modulation of the ventral tegmental area could prolong the behavioral changes. They found that intermittent acute patterned stimulation administered to the ventral tegmental area of Flinders sensitive line rats resulted in antidepressant-like and anti-anhedonic-like behaviors [96]. Strikingly, a study by Bregman et al. in 2018 found that the antidepressant-like e ffect of DBS was serotonin transporter-independent. This could be of benefit to some patients with a mutated serotonin transporter-promoter gene (5-HTTLPR), which underlies the poor response to conventional selective serotonin re-uptake inhibitors that target serotonin transporters [97].

Beside changes in neurochemical and neurotrophin profiles, neuroplasticity changes induced by DBS have also been investigated. For instance, Bambico et al. reported increased hippocampal neurogenesis and BDNF levels after vmPFC-DBS, which led to anti-anhedonic-like behaviors, but was not su fficient for an overall antidepressant-like e ffect [98]. Correspondingly, Liu et al. found a correlation between vmPFC-HFS and hippocampal neurogenesis and improvements in short- and long-term memory in middle-aged rats. This suggests that DBS has therapeutic potential in age-dependent memory deficits [99].

### *5.2. Other Brain Targets*

As preclinical studies have progressed, several brain targets of DBS have been established. Hamani et al. in 2014 demonstrated that DBS in the nucleus accumbens induced a similar antidepressant-like e ffect to DBS in the vmPFC, even though the stimulations modulated di fferent circuits. This may contribute to more customized stimulation targeting based on the patient's symptoms [100]. Bregman et al. reported that the HFS of the medial forebrain bundle induced antidepressant-like behaviors in the FST [101]. Interestingly, this antidepressant e ffect was not mediated by increases in either serotonin or dopamine release in the nucleus accumbens. Lim et al. in 2015 emphasized that only HFS of the vmPFC led to anti-anhedonic-like e ffects and these pronounced antidepressant-like e ffects were induced by modulating the activity of serotonergic neurons in the DRN [102]. However, the authors did not investigate the e ffects of di fferent stimulation parameters on depressive-like behaviors in various DBS targets. The study by Etiévant et al. supported the modulation of DRN by DBS and added that glial integrity was a prerequisite to the antidepressant-like outcome [103]. In another study, mice subjected to chronic social defeat stress were administered 7 days of 5-h vmPFC-DBS, which resulted in increased social interactive behavior accompanied by DRN modulation [104]. Interestingly, a recent study demonstrated that the potentiation of the anxiolytic response to vmPFC stimulation was associated with exposure to an enriched environment. This indicates that an enriched living environment can facilitate the beneficial e ffects of DBS intervention [105]. Creed et al. conducted DBS on the entopeduncular and the subthalamic nuclei to compare antidepressant-like e ffects [106]. Chronic Subthalamic nucleus DBS was reported to impair performance in the learned helplessness task, with no significant e ffects in anxiety tests. These results were associated with decreased hippocampal BDNF and TrkB mRNA. Interestingly, entopeduncular nucleus DBS did not increase depressive-like behavior in the learned helplessness task, indicating a superior target over the subthalamic nucleus for the treatment of depressive-like behaviors. Meng et al. reported reductions in depressive-like behaviors in animals stimulated in the lateral habenula; this observation was associated with elevations in dopamine, norepinephrine, and serotonin in both blood serum and in the hippocampus [107].

### *5.3. vmPFC-Linked Modulation of Other Structures*

Other structures have been found to be modulated by vmPFC-DBS. For example, Lim et al. reported that activation of the medial subthalamic nucleus contributed to antidepressant-like behavior [108]. In a rat model of post-traumatic stress disorder, IL-DBS reduced firing in the basolateral amygdala, which attenuated fear and produced a slight anxiolytic-like e ffect [109]. A recent study showed that DBS resulted in elevated spontaneous firing of noradrenergic locus coeruleus neurons and strengthened the coherence between the prefrontal cortex and locus coeruleus. The latter was protective against stress and was responsible for the antidepressant-like e ffect seen in FST [110]. On the other hand, Insel et al. reported that there was reduced communication between IL and ventral hippocampus in rats after 10 days of 8-h IL-DBS and such coherence was higher in depressed subjects [111]. Jiménez-Sánchez et al. in 2016 reported two studies on acute IL-DBS in naive and olfactory bulbectomized rat models. In naive animals, IL-DBS induced antidepressant-like behaviors and increased prefrontal glutamate efflux, which activated the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) to modulate DRN output [81]. In olfactory bulbectomized rats, similar changes were noted in the prefrontal serotonergic and glutamatergic output with the activation of AMPAR and antidepressant-like behaviors [33].

### *5.4. Synergism with Other Treatments*

Antidepressant-like e ffects in di fferent DBS paradigms are leading to some advancements in the field. One such investigation by Laver et al. in 2014 examined the use of augmentation agents such as buspirone, risperidone, and pindolol to enhance DBS e fficacy. However, these agents did not increase the antidepressant response of the rats receiving DBS treatment, when compared to those co-administered monoamine oxidase inhibitors in previous studies [64,112]. It is possible that a response may become evident in clinical trials. Perez-Caballero et al. in 2014 reported an interesting early response to stimulation, in which sham-treated rats had reduced immobility and increased swimming in FST at weeks 1 and 2, but not at week 6 post treatment. They reasoned that this was caused by insertion-induced inflammation as pretreatment by indomethacin reduced the expression of pro-inflammatory mediators (TNFα, COX1, COX2) and reversed the antidepressant-like behaviors in sham-treated animals [59]. Rummel et al. in 2016 reported that chronic continuous HFS did not have more benefits than chronic intermittent stimulation in treatment-resistant rats with congenitally learned helplessness [113].

### *5.5. Other Biological Parameters Modulated*

Similar to the research direction of clinical studies, preclinical studies have also attempted to characterize other biological parameters of DBS, including more precise electrode implantation. Lehto et al. characterized real-time fMRI responses in the brain following DBS, and found strong connectivity between the vmPFC and amygdala, which validated vmPFC as a target region [114]. Perez-Cabalerro et al. used PET scans to study the immediate e ffects of electrode implantation. They found that metabolism was decreased locally (vmPFC), but was increased in ventral regions, including dorsal and ventral hippocampus, piriform and insular cortex, nucleus accumbens, ventral tegmental area, ventral pallidum, hypothalamus, and the preoptic area [115]. This was in agreemen<sup>t</sup> with other studies on the e ffect of DBS on depressive-like behavior, but it is noteworthy to see these e ffects simply via electrode insertion.


**Table 3.** A list of preclinical studies on deep brain stimulation of the medial prefrontal cortex in rodents.






### *J. Clin. Med.* **2020**, *9*, 3260



CUS-sham animals.









Preclinical studies have progressed from studying the behavioral e ffects of DBS to understanding the accompanying cellular and molecular changes, be they local or distal nodes in the neurocircuitry. However, issues concerning the rodent homologs of SCC and the e ffect of stimulation in the subdivisions of vmPFC have ye<sup>t</sup> to be resolved and are discussed in the later part of the review.

### **6. Potential Mechanisms of Stimulation-Induced Antidepressant-Like Activities**

Several preclinical studies reported that DBS modulates neuronal activities in di fferent brain regions, leading to antidepressant-like behaviors (Figure 1A). The network-wide cellular and molecular changes caused by vmPFC-DBS can be classified into neuroplasticity-dependent and -independent changes (Figure 1B). Neuroplasticity-dependent e ffects included neurogenesis, increased synaptic plasticity, enhanced neurotrophin signaling, and potential activation of glial cell-mediated changes, whereas neuroplasticity-independent e ffects included changes in serotonergic (5-HT) and glutamatergic neurotransmission patterns, either locally or in distal structures. Other changes outside the scope of this review might also be relevant.

**Figure 1.** (**A**) Changes in local and distal neuronal activity after electrical stimulation of the ventromedial prefrontal cortex. (**B**) Neuroplasticity-dependent and -independent changes in di fferent structures following vmPFC-DBS. Abbreviations: AH, anterior hypothalamus; AMPAR, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor; BDNF, brain-derived neurotrophic factor; BLA, basolateral amygdaloid nucleus; Cg1,2, cingulate gyrus area 1, 2; CM, centromedial thalamic nucleus; DG, dentate gyrus; DMH, dorsomedial hypothalamus; DRD, dorsal raphe nucleus, dorsal part; DRVL, dorsal raphe nucleus, ventrolateral part; IntMC, interposed cerebellar nucleus, magnocelluar part; LA, lateral amygdaloid nucleus; LEnt, lateral entorhinal cortex; LHb, lateral habenula; MD, mediodorsal thalamic nucleus; mPFC, medial prefrontal cortex; OrbF, orbitofrontal cortex; PaMP, paraventricular hypothalamic nucleus, medial parvicellular; Pir, piriform cortex; PrL, prelimbic cortex; TeA, temporal association area; and 5-HT, serotonin.

### **Neuroplasticity-Dependent E** ff**ects of Electrical Stimulation**

### *(i) Neurogenesis is a Long-Term Cellular Change Brought About by Electrical Stimulation*

Post-mortem studies, pharmacological analyses, and electroconvulsive therapy reports have led to the neurogenic hypothesis of the pathogenesis of depression, whereby atrophy and apoptosis of hippocampal neurons correlated with depression and neurogenesis induce antidepressant-like effects [120,121]. As CA1 and subiculum in the hippocampus project substantially to the IL and the latter feeds back to the hippocampus via the relay nucleus reuniens in the thalamus [102,122], vmPFC-DBS induces a corollary of hippocampal neuromodulation that may mediate the antidepressant-like outcome. Etiévant et al. found that there was increased neurogenesis in the dentate gyrus of the dorsal and ventral hippocampus in rodents after 1-h IL-DBS, as detected by positive BrdU cells, and this was accompanied by reduced immobility in FST [103]. Similarly, Liu et al. reported proliferation of neuronal progenitors after chronic vmPFC-DBS, as demonstrated by increased positive BrdU and Dcx cell counts, as well as upregulated expressions of genes related to neurogenesis (NeuN, Syn, Dcx, Nes) and neuronal di fferentiation and protective functions (Angpt2, S100a4). These results were correlated with enhanced memory function, which may serve as another indication of vmPFC-DBS [99]. Bambico et al. confirmed that new cells with mature neuronal phenotype were found in the hippocampus after vmPFC-DBS, as detected by BrdU and NeuN co-expression. They also reported that temozolomide-induced reduction of these cells led to a longer latency to feed in a novelty-suppressed feeding test, but did not significantly change immobility in FST. This prompted the authors to further examine the anxiolytic-like and anti-anhedonic-like e ffects of vmPFC-DBS. In contrast, Winter et al. showed that 1 h of vmPFC-DBS with established DBS parameters in rodents did not increase the percentage of BrdU and Dcx double-stained cells in the dentate gyrus compared to the control [123]. Although research on the interaction between neurogenesis and substrates such as serotonin is ongoing, BDNF may be required to exert this antidepressant-like e ffect [98]. Neurogenesis is a widely investigated mechanism of DBS and these results indicate positive e ffects in the hippocampal region.

### *(ii) Synaptic Plasticity is Altered More Rapidly by Electrical Stimulation than by Neurogenesis*

Disruption in synaptic functions and signaling are implicated in the pathophysiology of MDD, considering their importance in neurotransmission and ultimately, in cell survival [20]. Chronic stress, a risk factor of MDD, was shown to cause retraction of dendrites in the medial prefrontal cortex [124] and CA3 of the hippocampus [125] in rodents. In the latter, the long-term potentiation (LTP) of synapses was compromised, a ffecting long-term memory formation [126]. Regarding the changes in synaptic plasticity caused by vmPFC-DBS, Liu et al. reported denser secondary dendritic spines in the dentate gyrus, as demonstrated by upregulated Syn expression correlated with Nes and Dcx. The authors also reported a slight (1.2 fold) increase in hippocampal BDNF gene expression, a regulator of synaptic plasticity [99]. More recently, Bezchlibnyk et al. found that 1 h of IL-DBS resulted in longer dendritic length and branch points localized in the basal and apical regions of hippocampal CA1 neurons, respectively. These results indicated that the acute stimulation stressed the indispensable connections between the hippocampus and vmPFC, which may have implications in MDD and its treatment [127].

Chakravarty et al. found that 5 days of 6-h vmPFC-DBS daily in 9-week-old C57Bl/6 mice resulted in a larger hippocampal volume and increased hippocampal synaptic density, as indicated by upregulated synaptophysin expression, a presynaptic marker [128]. Similarly, Veerakumar et al. found that chronic vmPFC-DBS in transgenic mice increased dendrite length and complexity of the 5-HT DRN neurons and upregulated the expression of postsynaptic markers synaptophysin and PSD-95 [104]. Moreover, Etiévant et al. reported synaptogenesis in the DRN, as indicated by higher expressions of PSD-95 and synapsin. This may explain the prolonged DRN neuronal activation during and after vmPFC-DBS, leading to an antidepressant-like e ffect [103]. According to earlier reviews, dendritic spines can respond swiftly and provide surfaces for synapse formation [126,129]. Given the

more dynamic properties of synapses compared to neurogenesis, synaptic plasticity may serve as an early indicator of vmPFC-DBS e fficacy. More preclinical studies characterizing the dynamics of synaptic plasticity under vmPFC-DBS are anticipated.

### *(iii) Neurotrophin Signaling Underlies the Antidepressant-Like E*ff*ect of Electrical Stimulation*

The neurotrophin BDNF is important in synaptic regulation, neuronal survival, and di fferentiation of new neuron terminals in the adult brain [130–132]. Preclinical studies reported that depressive-like rats subjected to chronic unpredictable stress [92,98] or olfactory bulbectomy [33] had lower BDNF levels, whereas DBS increased BDNF levels, thus preventing the development of depressive-like behaviors. Extracellularly, pro-BDNF is cleaved by tissue plasminogen activator/plasmin to form mature BDNF. The high-a ffinity tropomyosin-related kinase B (TrkB) receptor is activated by BDNF [133], leading to downstream phosphorylation of kinases, including protein kinase B (Akt) and extracellular signal-regulated kinases (ERK), which are important mediators of anti-apoptosis and proliferation, respectively [134]. Moreover, BDNF-TrkB triggers Serine 133 phosphorylation of transcription factor cAMP response element binding (CREB), leading to the formation of the dimer. The phosphorylated CREB dimer forms a larger transcriptional complex and alters multiple gene expressions including BDNF itself [135]. Encouragingly, Jiménez-Sánchez et al. showed that IL-DBS administered to olfactory bulbectomized rats activated these signaling pathways, as demonstrated by lowered Akt/pAkt, ERK/pERK, and CREB/pCREB ratios during 1 h of stimulation that increased again after stopping the stimulation, which was similar to the expression pattern of BDNF [33]. Further molecular studies are needed to characterize the action of vmPFC-DBS toward di fferent targets in this signaling cascade.

### *(iv) Potential Involvement of Glial Cells in Mediating the Outcome of Electrical Stimulation*

Glial cells may be involved in the pathogenesis of depression, as revealed by post-mortem studies of MDD patients, which found lower densities in the prefrontal cortex and amygdala, but increased levels in the hippocampal hilus [136–138]. The latter may be activated as a result of neuronal injury and decreasing neuronal populations [139,140]. Glial cells metabolically support neurons and regulate glutamate synthesis and thus, regulate synaptic plasticity. They may be modulated by DBS to potentiate the therapeutic e ffects [136]. This mechanism was supported in a study by Etiévant et al., which found that glial lesion by L-alpha-aminoadipic acid injection diminished antidepressant-like behaviors, hippocampal neurogenesis, and LTP induced by IL-DBS [103]. These findings led to the hypothesis that the neuronal-glial relationship is a determinant of the antidepressant-like e fficacy of DBS, but this requires further study. Perez-Caballero et al. also studied the e ffects of electrode implantation and analgesic supplements [115]. They found that implantation with non-NSAID analgesic treatments, like tramadol and morphine, did not ameliorate the anti-depressant e ffects of the electrode implantation. This observation was accompanied by an increase in glial marker GFAP-positive cells. This finding suggests that the supplementation of non-NSAIDs postoperatively could improve the comfort of patients.

### **Neuroplasticity-Independent E** ff**ects of Electrical Stimulation**

Besides modulating neuroplasticity-dependent mechanisms, DBS may manipulate some neuroplasticity-independent pathways to induce antidepressant-like e ffects. In a chronic mild stress model, depressive-like behaviors developed without significant deterioration of hippocampal neurogenesis or neuronal survival [141]. There are two likely inter-related neurotransmission systems that potentiate DBS e fficacy, namely serotonergic and glutamatergic systems.

### *(i) An Alternative Action of the Serotonergic System by Electrical Stimulation*

Results from preclinical studies have established an important role of the vmPFC-DRN axis and downstream 5-HT neurotransmission in the treatment of depression. Hamani et al. first reported that 5-HT neurotransmission was augmented by DBS, as shown by a four-fold increase in hippocampal

5-HT after 1 h of vmPFC-DBS [91]. The authors also suggested a relationship between the integral 5-HT system and DBS efficacy, as 5-HT depletion induced by DRN lesions with 5,7-dihydroxytryptamine injection diminished the antidepressant-like effects of vmPFC-DBS [91,92]. Similarly, a study by Perez-Caballero et al. showed that the administration of para-chlorophenylalanine ester impeded 5-HT biosynthesis and diminished the antidepressant-like behaviors in early DBS among IL sham-treated animals [59]. Interestingly, Volle et al. showed that DBS and fluoxetine could rescue the 5-HT system via different mechanisms [119]. Both treatments increased the amount of 5-HT at the end of the chronic treatments, but chronic fluoxetine treatment was associated with decreased expression of 5-HT1A in the prefrontal cortex and the hippocampus, whereas chronic DBS increased 5-HT1B expression in the prefrontal cortex, globus pallidus, substantia nigra, and raphe nuclei.

A study by Veerakumar et al. in a transgenic mouse model of chronic social defeat stress revealed normalization of 5-HT neuron excitability in DRN after vmPFC-DBS [104]. Moreover, Jiménez-Sánchez et al. found increased prefrontal 5-HT efflux after 1 h of IL-DBS in olfactory bulbectomized rats [33] and in naive rats [81]. Etiévant et al. also found spontaneous DRN 5-HT neuron activity increased with IL-DBS [103]. Strikingly, an electrophysiological study performed by Srejic et al. showed that IL-DBS decreased the firing rate of DRN neurons, including serotonergic subtypes via the activation of GABAergic interneurons and possibly by the inhibition of excitatory glutamatergic neurons that modulate the firing of 5-HT [142]. Hence, the positive response to DBS could be enhanced by more selective targeting of the neuronal population by pharmacological adjuncts or coupling with optogenetic techniques. A study by Bregman et al. in 2018 using a serotonin transporter knockout mouse model found that DBS increased hippocampal 5-HT concentration, despite mice responding poorly to fluoxetine, a conventional selective serotonin reuptake inhibitor that acts on serotonin transporter [97]. These findings revealed a novel antidepressant-like activity of DBS involving the 5-HT system primarily in the DRN [79].

### *(ii) Glutamatergic Neurotransmission is a Promising Target of Electrical Stimulation*

Jiménez-Sánchez et al. showed that there was enhanced prefrontal glutamatergic efflux together with changes in the local 5-HT profile [33,81]. The administration of AMPAR agonist and antagonist and subsequent FST showed that the increased glutamate led to antidepressant-like behaviors in animals [81]. The authors also found increased synthesis of the GluA1 subunit of AMPAR and postulated that their postsynaptic membrane insertion may explain the antidepressant-like outcome after 1 h of IL-DBS [33]. The activated glutamate output from the medial prefrontal cortex and frontal cortex enhanced 5-HT neuronal firing in the DRN [143], resulting in the antidepressant-like effect. However, Etiévant et al. argued that their activation was attributed to increased synaptogenesis in the DRN as previously described [103]. Nevertheless, Lim et al. hypothesized that a glutamatergic projection from the vmPFC to the medial subthalamic nucleus [144] may account for the antidepressant-like effects of vmPFC-DBS, as seen by increased c-Fos-immunoreactive cells in the medial subthalamic nucleus, increased sucrose consumption, and reduced immobility duration in FST [108]. With the emergence of glutamate-targeting pharmacotherapy [81], the ability to modulate glutamatergic transmission of DBS would add to the therapeutic novelty.

### **7. Concerns and Limitations of the Electrical Stimulation Studies**

The small sample sizes in several clinical studies might compromise the credibility of the DBS efficacy, even in studies with similar recruited DBS subjects or consistent outcomes [18,56]. Most of the clinical studies were open-label, which means the responses could be prone to the placebo effect, despite the early response characterized by Perez-Caballero [59]. <sup>E</sup>fficacy of DBS treatment could be overestimated, unless countered by long stimulation, randomization, and blinding [18,145], such as double-blinded and sham-controlled studies [55]. A major criterion in preclinical studies is that they should mimic clinical studies. As it is unfeasible to stimulate animals for 24 h a day as in clinical designs [128], the scheduling of the stimulations and behavioral assessments will thus be relevant to the validity of the outcomes. Stimulation during behavioral tests will be most similar to clinical studies, but this may interfere with the physiological functions of the animals [79]. Besides, DBS is normally carried out in animals for relatively short periods and the e ffects might not correlate well with chronic stimulation [33]. Some preclinical studies were conducted in naive animal models and would not be compatible with clinical trials as TRD patients will be recruited exclusively in clinical settings [33,93]. Moreover, carry-over e ffects and lesion e ffects may interfere with the results in both settings. Carry-over e ffects refer to behavioral or neurochemical changes after DBS ceases. This needs to be counteracted by a washout period to allow the subjects to resume their baseline physiological states before the next stimulation [79]. Lesion e ffects occur where responses are observed after electrode implantation [19,60,72]. This needs to be di fferentiated from true responses observed in preclinical studies by sham-treatment [67], otherwise, the therapeutic e ffect will be over-estimated. Generally speaking, care must be taken in the design of experiments and data analysis of preclinical studies to increase their translational value to clinical studies.

### **8. Prospective Approaches to Enhance Deep Brain Stimulation Safety and E** ffi**cacy**

Clinical response to SCC-DBS and various predictors can facilitate precise patient selection and customize the stimulation targets, thereby yielding maximal therapeutic outcomes with minimal adverse e ffects. For example, a lower baseline frontal theta cordance and incremental increase in the early stage of DBS indicates a clinical response [63]. E fforts have been made toward a more standardized approach to localize SCG in DBS responders [70]. Recently, real-time recording of the local field potential at the site of electrode implantation coupled with electroencephalogram have revealed network-wide clinical changes in DBS, which may improve the surgical precision [146]. Tractography-guided localization of electrodes, being more customized and precise, can improve the response rate [49]. A rechargeable DBS system should also be considered for long-term stimulation to reduce the need for surgery to replace batteries [17]. Lastly, given the high cost and invasiveness of DBS, more stringent regulation and evidence from randomized controlled studies are necessary to justify the benefits in TRD patients [147,148].
