Recording the Brain in Vivo: Emerging Technologies for the Exploration of Mental Health Conditions

Standfirst

Mounting interest in mental health conditions over the last two decades has been coupled with the increasing sophistication of techniques to study the brain in vivo.

Graphical Abstract

Keywords: :

• electrophysiology
• fiber photometry
• mental health conditions
• mental health disorders
• miniscopes
• neuropixels

The hunt for solutions to the multitude of issues that arise as populations age in numerous countries around the world, encapsulates a wide range of fields from healthcare to economics. In the field of neuroscience, this has led to a huge investment in the investigation of neurodegeneration and the aging brain, bringing with it advances in our understanding and treatment of conditions such as Alzheimer’s and dementia. However, the incidence of another class of conditions has also been growing in global populations, particularly in the West [1].

Mental health disorders, such as major depressive disorder (MDD) and substance use disorder (SUD), have only begun to receive adequate scientific attention in the last few decades. As such, pharmaceutical interventions for mental health disorders remain scarce and during the 30 years following the approval of the first selective serotonin reuptake inhibitors (SSRIs) for the treatment of MDD in 1988, very little has changed [2]. Subsequent therapeutics mostly focus on improvements to the compounds’ safety profiles and target similar signaling pathways in the brain.

However, as these disorders, and our awareness of them, become more prevalent, research efforts to address them have become more commonplace; as a result, our understanding of the molecular basis underlying these conditions has dramatically improved. Furthermore, since an initial proof-of-concept study displayed the positive performance of the hallucinogenic anesthetic ketamine in the treatment of MDD, the glutamatergic system has been increasingly pursued as a therapeutic target for the condition [3]. This has led to the approval of two new therapeutics by the FDA in recent years: esketamine for treatment-resistant depression [4] and brexanolone for postpartum depression [5], both of which were approved in 2019 and have a distinct mechanism of action from SSRIs.

These developments have arisen amongst a reemergence in the study of hallucinatory psychoactive substances. One such substance, psilocybin, was last year approved for use in the treatment of PTSD in Australia and has successfully progressed through two Phase II clinical trials for its use in the treatment of MDD in the USA and UK [6,7].

As ever, technological advancements have underpinned our understanding of these conditions, with new technologies emerging throughout different areas of investigation, from the observation techniques used in top-level behavioral studies to the spatial multiomic techniques implemented in the investigation of the biochemical pathways involved in these conditions. One application in particular that has seen huge advancements in the last two decades is our ability to monitor the neural activity of brains in vivo as living organisms interact and freely behave. Here we explore these emergent methodologies and how they are transforming research into mental health disorders.

One of the improvements in the conduct of animal model behavioral studies in the last few decades has been the ability to monitor the neural activities of animal models as they interact or conduct a task. The development of increasingly miniscule imaging methods and high-density electrophysiological recording techniques are the two main methods by which this has been made possible.

Miniscopes

The first head-mounted two-photon microscope was developed in 2001 [8]. Weighing in at 25 g and measuring 7.5 cm in length, the initial microscope (Figure 1) was connected to the excitation laser and optical setup by a 2 m-long tether and was designed for use in rats. This was a major step forward in the study of the brain at the time – overcoming the requirement to fix animal models at the head for imaging. Fixation presented several problems: it was impossible to observe the neural activity of an organism as it behaved naturally and produced further ethical and practical confounds as it could induce stress in the animal.

Figure 1. Schematic of (A) the original head-mounted two-photon microscope setup and (B) the technology.

Redrawn with permission from Helmchen et al. [[8]; published under an Elsevier user license.

However, there were some essential limitations with the early versions of the miniscope. First, the weight and bulkiness of the technology limited its use primarily to rats and larger animals an could still impact the behavior of the animal. The depth of field and field of view were also restrictive. Since its publication, however, each of these limitations has been sufficiently addressed to render these technologies suitable for wider use by neuroscientists [9].

Contemporary miniscopes can weigh as little as 1 g [10] and provide a field of view of 8 × 10 mm [11]. In addition to improving the capability of the technology, the decreased size has expanded their compatibility to mice, which have a wider range of available genetic tools with which to conduct investigations than rats.

In a recent example of the utility of these technologies, a team of researchers from the University of California, Los Angeles (CA, USA), utilized miniscopes to observe medium-sized spiny neurons (MSNs) and cortical pyramidal neurons in free-moving mice in order to determine the impact of oxycodone on their activity. Oxycodone is the opioid, known by its brand name as Oxycontin, at the center of the opioid crisis in the USA. MSNs had previously been indicated to play a role in the acquisition of habitual opioid addiction [12].

In a procedure typical to the application of miniscopes, the dorsal striatum of the mice in the study was transfected with the calcium indicator GCaMP6f via injection of an adeno-associated viral (AAV) vector. A total of 2 weeks subsequent, surgery was conducted to insert a gradient index micro (GRIN) lens into the section of the murine cortex above the dorsal striatum before the miniscope was mounted above it. Using this setup the team was able to visualize the activity of the MSNs and cortical pyramidal neurons after the administration of oxycodone [13].

The team observed that post oxycodone administration, an increase in the motility of the mice was coupled with a decreased number of active MSNs while activity in cortical pyramidal neurons increased. Coupling these investigations with in vitro electrophysiological recordings and Ca²⁺ imaging of brain slices, the team was able to conclude that the activation of μ-opioid receptors by oxycodone “hampers the communication along corticostriatal and thalamostriatal pathways, the main excitatory inputs onto MSNs, probably via presynaptic inhibition of glutamate release.“ This provides a potential mechanistic explanation for the involvement of these neurons in addiction. Meanwhile, the increased frequency in the activation of projection neurons of the cerebral cortex suggested an “important role in behavioral activation” [12].

In a study published the following year regarding alcohol addiction, researchers from the University of Tennessee (TN, USA) were able to simultaneously observe fluctuations in neuronal activity and microvessel dilation in response to ethanol administration in mice using a dual color miniscope, demonstrating its suitability for use in studies of alcohol addiction [13].

The same year an international collaboration used in vivo miniscope recordings in combination with mass spectrometry, protein analysis and electrophysiological analysis to expose a potential cellular and biomolecular mechanism behind deacetylase sirtuin 1’s (SIRT1) association with anxiety in a mouse model. These results could provide a novel therapeutic target for future anxiety medications [14].

Fiber photometry

Fiber photometry is a similar technique, using implanted optic fibers rather than a microscope to direct excited light to specific brain regions that have been prepared with genetically encoded fluorescent indicators (GEIs). These indicators provide a fluorescent readout for general or subtype-selective neural activity, which is collected by the optic fiber and returned to a photodetector and amplifier for signal processing and analysis [15].

First developed in 2005, this technology has also seen improvements in size and practicality; however, the main development that has popularized this technique is the recent emergence of novel fluorescent sensors for a far wider range of molecules – such as extracellular ligands – than was previously available [16]. Additionally, when it comes to the surgery required to embed this kind of technology, fiber photometry has the added benefit of being less invasive than miniscopes, reducing their potential influence on animal behavior.

Fiber photometry provides a measure of bulk neuronal activity as opposed to the cellular resolution provided by miniscopes. However, their simplicity allows multiple fibers to be implanted into different areas of the brain for simultaneous deep-brain investigations. This makes them useful for the investigation of disparate brain regions and how they work in concert during a particular behavior, even if they are not physically connected [16].

In a recent study, researchers from the University of Fr eiburg (Freiburg im Breisgau, Germany) applied fiber photometry to investigate the mechanism underlying the therapeutic effect of an emerging potential treatment for depression: deep brain stimulation (DBS). DBS has been approved for use in other psychiatric disorders, such as obsessive-compulsive disorder; in previous clinical trials, the stimulation of the superolateral branch of the medial forebrain bundle (slMFB) with a DBS electrode has been shown to have fast-acting and lasting relief for treatment-resistant depression. However, the mechanism behind this relief is not understood [15].

A current theory for the pathway underlying this therapeutic effect is that DBS of the slMFB helps to modulate the activity of neurons in the reward pathway, which is dysfunctional in MDD, ultimately influencing dopamine activity in the nucleus accumbens (Nac). To test the validity of this hypothesis, the team injected the brains of three rats, targeting the Nac, with a novel GEI, GRAB_DA2m, which binds to dopamine receptors and fluoresces with increased intensity when they are bound by dopamine. The rats each had DBS electrodes implanted in their MFB and optical fibers implanted in their Nac.

The team observed the impact of DBS of the MFB on the release of dopamine in the Nac after the animals received stimulation significant enough to induce SEEKING behavior. The SEEKING behavioral response is displayed by an increase in explorative, searching and rearing behavior and can be considered a readout for a positive emotional state, standing in as a useful proxy for therapeutic effect in this study. DBS was administered for 5 or 20 seconds over the course of 14 days. The team ultimately revealed that DBS led to an increase in the dopamine activity in the Nac, peaking 1 second into stimulation. This showed that DBS leads to increased activity of dopamine in the Nac and contributes to the existing theory for its therapeutic effect.

This study highlights the utility of fiber photometry as a primary technology around which to design a study. However, it is equally adept as a supporting technology, as highlighted by Gui-Ying Zan et al. from the Chinese Academy of Sciences (Shanghai, China), who used fiber photometry as a more traditional Ca²⁺ activity recording method to determine the activity of neurons in the Nac during an investigation of the pathways underlying the link between depression and opioid abstinence following a period of addiction [17].

In this study, the team used a suite of techniques, including immunohistochemistry, fiber photometry, gene expression analysis and patch clamp recordings, to determine that during opioid abstinence, there is an increase in the expression of the protein dynorphin in the amygdala, which activates k-opioid receptors, ultimately reducing the excitation of the Nac, the fundamental cause of the abstinence-induced depression-like behavior.

Neuropixels

The recent developments in imaging methods that allow for the monitoring of neural activity and the dynamics of a host of different neurotransmitters and compounds have undoubtedly been groundbreaking. However, one essential challenge yet to be overcome is the development of an approach to image the neural activity of the human brain in vivo. Numerous ethical issues present themselves that prove prohibitive, and one can imagine that securing study participants to volunteer for a section of their skull to be replaced with an optical lens could prove to be considerably challenging.

An alternative to this approach that has long been utilized in studies of the in vivo human brain has been electrophysiology. Electrophysiological techniques traditionally involved the use of electrode grids placed across the top of the cortex that provide an insight into the electrical activity in different regions of the brain on a macro level by recording field potentials at the surface of the brain, which is a technique known as electrocorticography. Alternative approaches to electrocorticography utilized extracellular probes that could record the activity of neurons; however, until recently these probes could only collect data from a limited number of neurons at the same time [18].

In 2017, an international collaboration led by the Howard Hughes Medical Institute Janelia Research Campus (VA, USA) invented Neuropixels, which are silicone probes containing over 1000 recording sites. In the initial paper documenting Neuropixels’ development, the authors revealed that when used in tandem, two Neuropixels were capable of collecting data from 500 neurons in five distinct regions of the mouse brain [18].

A later update to this probe came in 2021 with Neuropixels 2.0, which are a third of the size of their predecessor, yet contain more recording sites closely packed together, which gives then the spatial sensitivity to continue recording information from a single neuron as it moves across the probe. What’s more, the new probes contain four shanks that enter the brain instead of one, enabling recordings to be taken from a wider section of the brain. These probes, in particular, are capable of tracking thousands of individual neurons in the brains of animal models over a prolonged period of time.

As with miniscopes and fiber photometry, there are clear applications for the use of Neuropixels 2.0 in behavior studies of mental health conditions in animal models and they have indeed been applied in this area [19]. However, since their invention, researchers have also been enamored by their potential use in humans due to their uniquely practical attributes. In 2023, the first-in-human use of Neuropixels was demonstrated by a Boston-based collaboration led by Sydney Cash and Angelique Paulk of Harvard Medical School (MA, USA), who successfully and safely inserted the probe into the brain of patients undergoing resection surgeries for the management of epilepsy and tumors and for the insertion of DBS electrodes for patients with Parkinson’s [20].

A few months later, a collaboration between researchers at the University of California, San Francisco (UCSF; CA, USA), and the University of Columbia (NY, USA), led by Edward Chang (UCSF) reported the use of Neuropixels to record 685 neurons spread across nine sites in different cortical layers of an auditory brain region critical to speech. Recordings were taken as participants listened to spoken sentences and enabled the team to observe individual neurons encoding different sound cues such as vowels and consonants alongside many other aspects of speech. What’s more, when neural activity recordings were assessed across all cortical levels, they were found to be predictive of results found via electrocorticography, enabling researchers to determine the neural pathways and activity underlying electrophysiological results taken from the surface of the brain [21].

Speaking on a recent episode of the Talking Techniques Podcast, Michael Long of the New York University Grossman School of Medicine (NY, USA) revealed that these adapted Neuropixels have “been used to astonishing effect,” by researchers such as Chang and Cash. Any risk of breakage has been largely minimized by inserting the probes into areas that are going to be removed to complete the primary goal of the neurosurgery [22].

Additionally, Long has helped to validate an alternate approach preferred by his lab for use in humans. In this configuration, manufactured by Diagnostic Biochips, high-density electrodes capable of recording spiking activity are placed on a flexible metal electrode that can safely be introduced to the brain in several neurosurgical contexts, enabling him and his colleagues at the University of Iowa “to perform our recordings with an instrument that moves with the brain in a completely safe environment.” So far, Long has been able to use this approach in seven patients, inserting them prior to, and in the same path as, a much larger DBS electrode, to ensure minimal risk to the patient.

While these developing probes and their use in humans are in their infancy, specific examples of their use in the investigation of mental health disorders are few and far between, but their potential to impact the field is evident.

The emergent wave of interest in mental health and novel therapeutic approaches, alongside the increasing variety of angles from which researchers are examining mental health disorders, have now been matched with a suite of new and developing technologies with which to explore them. As the challenges surrounding mental health disorders grow, it seems the world of research is rising to meet them.