Transnasal targeted delivery of therapeutics in central nervous system diseases: a narrative review

Currently, neurointervention, surgery, medication, and central nervous system (CNS) stimulation are the main treatments used in CNS diseases. These approaches are used to overcome the blood brain barrier (BBB), but they have limitations that necessitate the development of targeted delivery methods. Thus, recent research has focused on spatiotemporally direct and indirect targeted delivery methods because they decrease the effect on nontarget cells, thus minimizing side effects and increasing the patient’s quality of life. Methods that enable therapeutics to be directly passed through the BBB to facilitate delivery to target cells include the use of nanomedicine (nanoparticles and extracellular vesicles), and magnetic field-mediated delivery. Nanoparticles are divided into organic, inorganic types depending on their outer shell composition. Extracellular vesicles consist of apoptotic bodies, microvesicles, and exosomes. Magnetic field-mediated delivery methods include magnetic field-mediated passive/actively-assisted navigation, magnetotactic bacteria, magnetic resonance navigation, and magnetic nanobots—in developmental chronological order of when they were developed. Indirect methods increase the BBB permeability, allowing therapeutics to reach the CNS, and include chemical delivery and mechanical delivery (focused ultrasound and LASER therapy). Chemical methods (chemical permeation enhancers) include mannitol, a prevalent BBB permeabilizer, and other chemicals—bradykinin and 1-O-pentylglycerol—to resolve the limitations of mannitol. Focused ultrasound is in either high intensity or low intensity. LASER therapies includes three types: laser interstitial therapy, photodynamic therapy, and photobiomodulation therapy. The combination of direct and indirect methods is not as common as their individual use but represents an area for further research in the field. This review aims to analyze the advantages and disadvantages of these methods, describe the combined use of direct and indirect deliveries, and provide the future prospects of each targeted delivery method. We conclude that the most promising method is the nose-to-CNS delivery of hybrid nanomedicine, multiple combination of organic, inorganic nanoparticles and exosomes, via magnetic resonance navigation following preconditioning treatment with photobiomodulation therapy or focused ultrasound in low intensity as a strategy for differentiating this review from others on targeted CNS delivery; however, additional studies are needed to demonstrate the application of this approach in more complex in vivo pathways

Reversible Plasticity of Fear Memory-Encoding Amygdala Synaptic Circuits Even after Fear Memory Consolidation

It is generally believed that after memory consolidation, memory-encoding synaptic circuits are persistently modified and become less plastic. This, however, may hinder the remaining capacity of information storage in a given neural circuit. Here we consider the hypothesis that memory-encoding synaptic circuits still retain reversible plasticity even after memory consolidation. To test this, we employed a protocol of auditory fear conditioning which recruited the vast majority of the thalamic input synaptic circuit to the lateral amygdala (T-LA synaptic circuit; a storage site for fear memory) with fear conditioning-induced synaptic plasticity. Subsequently the fear memory-encoding synaptic circuits were challenged with fear extinction and re-conditioning to determine whether these circuits exhibit reversible plasticity. We found that fear memory-encoding T-LA synaptic circuit exhibited dynamic efficacy changes in tight correlation with fear memory strength even after fear memory consolidation. Initial conditioning or re-conditioning brought T-LA synaptic circuit near the ceiling of their modification range (occluding LTP and enhancing depotentiation in brain slices prepared from conditioned or re-conditioned rats), while extinction reversed this change (reinstating LTP and occluding depotentiation in brain slices prepared from extinguished rats). Consistently, fear conditioning-induced synaptic potentiation at T-LA synapses was functionally reversed by extinction and reinstated by subsequent re-conditioning. These results suggest reversible plasticity of fear memory-encoding circuits even after fear memory consolidation. This reversible plasticity of memory-encoding synapses may be involved in updating the contents of original memory even after memory consolidation

ABA renewal involves enhancements in both GluA2-lacking AMPA receptor activity and GluA1 phosphorylation in the lateral amygdala.

Fear renewal, the context-specific relapse of fear following fear extinction, is a leading animal model of post-traumatic stress disorders (PTSD) and fear-related disorders. Although fear extinction can diminish fear responses, this effect is restricted to the context where the extinction is carried out, and the extinguished fear strongly relapses when assessed in the original acquisition context (ABA renewal) or in a context distinct from the conditioning and extinction contexts (ABC renewal). We have previously identified Ser831 phosphorylation of GluA1 subunit in the lateral amygdala (LA) as a key molecular mechanism for ABC renewal. However, molecular mechanisms underlying ABA renewal remain to be elucidated. Here, we found that both the excitatory synaptic efficacy and GluA2-lacking AMPAR activity at thalamic input synapses onto the LA (T-LA synapses) were enhanced upon ABA renewal. GluA2-lacking AMPAR activity was also increased during low-threshold potentiation, a potential cellular substrate of renewal, at T-LA synapses. The microinjection of 1-naphtylacetyl-spermine (NASPM), a selective blocker of GluA2-lacking AMPARs, into the LA attenuated ABA renewal, suggesting a critical role of GluA2-lacking AMPARs in ABA renewal. We also found that Ser831 phosphorylation of GluA1 in the LA was increased upon ABA renewal. We developed a short peptide mimicking the Ser831-containing C-tail region of GluA1, which can be phosphorylated upon renewal (GluA1S); thus, the phosphorylated GluA1S may compete with Ser831-phosphorylated GluA1. This GluA1S peptide blocked the low-threshold potentiation when dialyzed into a recorded neuron. The microinjection of a cell-permeable form of GluA1S peptide into the LA attenuated ABA renewal. In support of the GluA1S experiments, a GluA1D peptide (in which the serine at 831 is replaced with a phosphomimetic amino acid, aspartate) attenuated ABA renewal when microinjected into the LA. These findings suggest that enhancements in both the GluA2-lacking AMPAR activity and GluA1 phosphorylation at Ser831 are required for ABA renewal

Inhibition of GluR2-lacking AMPAR activity in the LA is required for ABA renewal.

<p><b>A.</b> The behavioral procedure for the experiments. NASPM, a selective blocker of GluA2-lacking AMPARs, was microinjected 15 min before tone presentation in context C on Day 6. In this figure, a weaker conditioning protocol was used (see Methods for additional details). <b>B.</b> Microinjection of NASPM (40 µg) into the LA impaired ABA renewal relative to the microinjection of saline (F_2,23 = 10.44, p = 0.0006, one-way ANOVA; Saline, 66.9±7.0%, n = 12; 10 µg NASPM, 44.1±11.6%, n = 7; 40 µg NASPM, 16.0±3.5%, n = 7; p<0.001, Newman-Keuls post-test). <b>C.</b> Schematic representation of the injector cannula tips. Histological plates illustrating the injection site in the LA were adopted from the rat brain atlas <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0100108#pone.0100108-Paxinos1" target="_blank">[50]</a>.</p

The GluR1_S peptide inhibits both low-threshold potentiation and its associated enhancement in the RI.

<p><b>A.</b> Left: Inclusion of GluA1_S in the internal solution inhibited the low-threshold potentiation relative to GluA1_A (GluR1_A, 135.9±7.8% of baseline, n = 12; GluA1_S, 113.3±6.6% of baseline, n = 16). Representative traces are superimposed averages of the EPSCs before and 20 min after pairing. Scale bars, 20 ms and 50 pA. Right: The inclusion of GluA1_S or GluA1_A in the internal solution did not have a significant effect on basal synaptic transmission (GluA1s, 104.1±6.7% of baseline, n = 5, p>0.5; GluA1_A, 109.7±5.8 of baseline, n = 5, p>0.1; paired <i>t</i>-test). <b>B1</b>. The pairing protocol produced an enhancement in the RI and the AMPA EPSC amplitudes when cells were dialyzed with GluA1_A. Left: Sample traces from an individual experiment. Right: Summary data for the RI changes after the induction of low-threshold potentiation (114.9±5.9% of baseline, n = 13). NMDAR-meditated EPSCs did not change significantly after the induction of low-threshold potentiation (113.4±7.4% of baseline, p = 0.0930, paired <i>t</i>-test). <b>B2</b>. The inclusion of GluA1_S in the internal solution attenuated the RI increase associated with the low-threshold potentiation. Left, Sample traces from an individual experiment. Right, Summary data for the RI changes after the induction of low-threshold potentiation (103.9±4.5% of baseline, n = 13). NMDAR-meditated EPSCs did not change (106.9±4.6% of baseline, p = 0.1563, paired <i>t</i>-test). *, p<0.05.</p

The microinjection of a cell permeable form of the GluA1-derived peptides into the LA attenuates ABA renewal.

<p><b>A.</b> The behavioral procedure. The tone stimulus used for renewal was 60<b> </b>sec in duration on Day 6. In this figure, a weaker conditioning protocol was used (<i>see</i> Methods for additional details). <b>B1</b>. Upper, The microinjection of GluA1_S into the LA impaired ABA renewal relative to GluA1_A (GluA1_A, 41.43±2.68%, n = 8; GluA1_S, 21.24±5.63%, n = 9; p<0.01, unpaired <i>t</i>-test). Lower, Schematic representation of the injector cannula tips. Histological plates illustrating the injection site in the LA were adopted from the rat brain atlas <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0100108#pone.0100108-Paxinos1" target="_blank">[50]</a> (○, GluA1_A; •, GluA1_S). <b>B2</b>. Top, GluA1_A peptide injection had no effects on ABA renewal relative to vehicle controls (vehicle, 56.09±6.78%, n = 9; GluA1_A, 53.70±11.05%, n = 10; p>0.5, unpaired <i>t</i>-test). Bottom, Schematic representation of the injector cannula tips. Histological plates illustrating the injection site in the LA were adopted from the rat brain atlas (○, Vehicle; •, GluA1_A). <b>C.</b> Diffusion of the fluorescent dansyl-tat-GluR1_S peptide (1<b> </b>nmol) within 1<b> </b>h after the microinjection, as visualized with a multiphoton microscope (top). The white arrow indicates the end of the injector cannula. Peptide transduction in individual LA neurons at high magnification (bottom). Ce, central amygdala; BA, basal amygdala. <b>D1</b>. Top: Microinjection of GluA1_D into the LA impaired ABA renewal relative to GluA1_A (GluA1_A, 66.82±5.62%, n = 6; GluA1_D, 36.70±6.77%, n = 6; p<0.01, unpaired t-test). Bottom: Schematic representation of the injector cannula tips (○, GluA1_A; •, GluA1_D). <b>D2</b>. Top: GluA1_A injection had no effects on ABA renewal relative to vehicle controls (vehicle, 74.01±5.65%, n = 6; GluA1_A, 77.37±7.24%, n = 4; p>0.05, unpaired t-test). Bottom: Schematic representation of the injector cannula tips (○, Vehicle; •, GluA1_A).</p

ABA renewal-inducing stimuli produce a context-dependent enhancement of synaptic efficacy at T-LA synapses.

<p><b>A.</b> The behavioral procedure. On Day 7, brain slices were prepared immediately after a tone test (ABB-tone and ABA-tone group) or context exposure (ABC-context group). In unpaired controls, one set of rats was killed for the preparation of brain slices, while another set was monitored for conditioned freezing to a CS on Day 7. <b>B</b> and <b>C.</b> Pooled behavioral results. **, p<0.01. <b>D.</b> Input-output curves for EPSCs in unpaired controls (n = 7), ABC-context (n = 7), ABB-tone (n = 16) and ABA-tone groups (n = 9). The representative traces are the averages of four responses evoked by input stimulations of 35 µA. Scale bars, 20 ms and 100 pA.</p

Reversible modification of T-LA synaptic efficacy within a fixed modification range.

<p><b>A</b>. <b>Left</b>, Ceiling of the behaviorally modifiable range assessed with pairing-induced LTP. Robust LTP was observed in the groups for which T-LA synaptic weights were predicted to be at the floor of the range (naïve, extinction, unpaired group 1 & 2), whereas LTP was occluded in the groups for which T-LA synaptic weights were expected to be at the ceiling (conditioned, reconditioned). <b>Right</b>, Floor of the range estimated with depotentiation. Depotentiation was observed in the groups for which T-LA synaptic weights were predicted to be at the ceiling (conditioned, reconditioned), whereas depotentiation was absent in the groups for which T-LA synaptic weights were expected to be at the floor (naïve, extinction, unpaired group 1 and 2). <b>B.</b> Summary of the results shown in Fig. 2A. To avoid possible bias, the experiments in Fig. 2A were performed with the experimenter blind to the behavioral group.</p

ABA renewal-inducing stimuli enhance the amplitude of AMPAR-mediated mEPSCs at T-LA synapses.

<p><b>A.</b> The behavioral procedure. On Day 7, brain slices were prepared immediately after a tone test (ABA-tone group). In the extinction group, one set of rats was killed for the preparation of brain slices, while another set was monitored for conditioned freezing to CS on Day 7. <b>B.</b> Upper left: Sample traces of evoked EPSCs in the presence of Ca²⁺ or Sr²⁺. Scale bars, 50<b> </b>ms and 10 pA. Note that the representative traces were further processed with a digital Gaussian filter for better display. Lower left: Cumulative amplitude distributions of evoked mEPSCs in the presence of Sr²⁺ (n = 300 events per cell). Upper right: Mean amplitude of mEPSCs evoked in the presence of Sr²⁺ (F_2,16 = 5.896, p = 0.0121; naïve, 14.3±0.9 pA, n = 7; extinction,14.0±0.7 pA, n = 7; ABA-tone, 17.5±0.4 pA, n = 5; *, p<0.05). Lower right: Mean frequency of mEPSCs evoked in the presence of Sr²⁺ (naïve, 8.0±1.1<b> </b>Hz, n = 7; extinction, 7.4±1.2<b> </b>Hz, n = 7; ABA-tone, 9.4±1.7<b> </b>Hz, n = 5).</p

Behavioral procedures.

<p><b>A.</b> Schematic diagram for behavioral procedures. Conditioning was carried out for two consecutive days, followed by extinction (three days) and reconditioning (two days) in a distinct context. White and gray tones in the rectangles represent context A and B, respectively. On day eight, brain slices were acquired from rats, while a separate set of animals were tested for fear memory retention. <b>B.</b> Pooled behavioral results for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024260#pone-0024260-g002" target="_blank">Fig. 2</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024260#pone-0024260-g003" target="_blank">3</a>.</p