Repetitive transcranial magnetic stimulation (rTMS) is a form of non-invasive brain stimulation frequently used to induce neuroplasticity in the brain. Even at low intensities, rTMS has been shown to modulate aspects of neuronal plasticity such as motor learning and structural reorganisation of neural tissue. However, the impact of low intensity rTMS on glial cells such as astrocytes remains largely unknown. This study investigated changes in RNA (qPCR array: 125 selected genes) and protein levels (immunofluorescence) in cultured mouse astrocytes following a single session of low intensity repetitive magnetic stimulation (LI-rMS e 18 mT). Purified neonatal cortical astrocyte cultures were stimulated with either 1Hz (600 pulses), 10Hz (600 or 6000 pulses) or sham (0 pulses) LI-rMS, followed by RNA extraction at 5 h post-stimulation, or fixation at either 5 or 24-h post-stimulation. LI-rMS resulted in a two-to-four-fold downregulation of mRNA transcripts related to calcium signalling (Stim1 and Orai3), inflammatory molecules (Icam1) and neural plasticity (Ncam1). 10Hz reduced expression of Stim1, Orai3, Kcnmb4, and Ncam1 mRNA, whereas 1Hz reduced expression of Icam1 mRNA and signalling-related genes. Protein levels followed a similar pattern for 10Hz rMS, with a significant reduction of STIM1, ORAI3, KCNMB4, and NCAM1 protein compared to sham, but 1Hz increased STIM1 and ORAI3 protein levels relative to sham. These findings demonstrate the ability of 1Hz and 10Hz LI-rMS to modulate specific aspects of astrocytic phenotype, potentially contributing to the known effects of low intensity rTMS on excitability and neuroplasticity.

During mammalian visual system development, retinal ganglion cells (RGCs) undergo extensive apoptotic death. In mouse retina, approximately 50% of RGCs present at birth (postnatal day 0; P0) die by P5, at a time when axons innervate central targets such as the superior colliculus (SC). We examined whether RGCs that make short-range axonal targeting errors within the contralateral SC are more likely to be eliminated during the peak period of RGC death (P1-P5), compared with RGCs initially making more accurate retinotopic connections. A small volume (2.3 nL) of the retrograde nucleophilic dye Hoechst 33342 was injected into the superficial left SC of anesthetized neonatal C57Bl/6J mice at P1 (n 5 5) or P4 (n 5 8), and the contralateral retina wholemounted 12 hr later. Retrogradely labelled healthy and dying (pyknotic) RGCs were identified by morphological criteria and counted. The percentage of pyknotic RGCs was analyzed in relation to distance from the area of highest density RGC labelling, presumed to represent the most topographically accurate population. As expected, pyknotic RGC density at P1 was significantly greater than P4 (p < 0.05). At P4, the density of healthy RGCs 500-750 mm away from the central region was significantly less, although this was not reflected in altered pyknotic rates. However, at P1 there was a trend (p 5 0.08) for an increased proportion of pyknotic RGCs, specifically in temporal parts of the retina outside the densely labelled center. Overall, the lack of consistent association between short-range targeting errors and cell death suggests that most postnatal RGC loss is not directly related to topographic accuracy.

During development of retinofugal pathways there is naturally occurring cell death of at least 50% of retinal ganglion cells (RGCs). In rats, RGC death occurs over a protracted pre- and early postnatal period, the timing linked to the onset of axonal ingrowth into central visual targets. Gene expression studies suggest that developing RGCs switch from local to target-derived neurotrophic support during this innervation phase. Here we investigated, in vitro and in vivo, how RGC birthdate affects the timing of the transition from intra-retinal to target-derived neurotrophin dependence. RGCs were pre-labeled with 5-Bromo-2′-Deoxyuridine (BrdU) at embryonic (E) day 15 or 18. For in vitro studies, RGCs were purified from postnatal day 1 (P1) rat pups and cultured with or without: (i) brain derived neurotrophic factor (BDNF), (ii) blocking antibodies to BDNF and neurotrophin 4/5 (NT-4/5), or (iii) a tropomyosin receptor kinase B fusion protein (TrkB-Fc). RGC viability was quantified 24 and 48 h after plating. By 48 h, the survival of purified βIII-tubulin immunopositive E15 but not E18 RGCs was dependent on addition of BDNF to the culture medium. For E18 RGCs, in the absence of exogenous BDNF, addition of blocking antibodies or TrkB-Fc reduced RGC viability at both 24 and 48 h by 25–40%. While this decrease was not significant due to high variance, importantly, each blocking method also consistently reduced complex process expression in surviving RGCs. In vivo, survival of BrdU and Brn3a co-labeled E15 or E18 RGCs was quantified in rats 24 h after P1 or P5 injection into the eye or contralateral superior colliculus (SC) of BDNF and NT-4/5 antibodies, or serum vehicle. The density of E15 RGCs 24 h after P1 or P5 injection of blocking antibodies was reduced after SC but not intraretinal injection. Antibody injections into either site had little obvious impact on viability of the substantially smaller population of E18 RGCs. In summary, most early postnatal RGC death in the rat involves the elimination of early-born RGCs with their survival primarily dependent upon the availability of target derived BDNF during this time. In contrast, late-born RGC survival may be influenced by additional factors, suggesting an association between RGC birthdate and developmental death mechanisms.

Static magnetic stimulation (SMS) is a form of non-invasive brain stimulation that can alter neural activity and induce neural plasticity that outlasts the period of stimulation. While SMS is typically delivered for short periods (e.g., 10 minutes) to alter corticospinal excitability or motor behaviours, the plasticity mechanisms that can be induced with longer periods of stimulation have not been explored. In mammalian neurons, the axon initial segment (AIS) is the site of action potential initiation and undergoes structural plasticity as a homeostatic mechanism to counteract chronic changes in neuronal activity. Therefore, we investigated whether the chronic application of SMS would induce structural AIS plasticity in cortical neurons. SMS (0.5 Tesla in intensity) was delivered to postnatally derived mouse primary cortical neurons consisting of mainly inhibitory neurons, for 6 or 48 hours beginning from 7 days in vitro (DIV7). AIS structural plasticity (length and starting distance from the soma) was quantified immediately after and 24 hours post-stimulation. Following 6 hours of stimulation, we observed an immediate decrease in median AIS length compared to control, that persisted to 24 hours post stimulation. In addition, there was a distal shift in the AIS start position relative to the soma that was only observed 24 hours after the 6-hour stimulation. Following 48 hours of stimulation, we observed an immediate shortening of AIS length and a distal shift in AIS start position relative to the soma, however only the distal shift in AIS start position persisted to 24 hours post-stimulation. Our findings provide the foundation to expand the use of SMS to more chronic applications as a method to study or promote AIS plasticity non-invasively.