Neural recordings can differentiate between spontaneously metastasizing melanomas and melanomas with low metastatic potential.

Multiple studies report that melanomas are innervated tumors with sensory and sympathetic fibers where these neural fibers play crucial functional roles in tumor growth and metastasis with branch specificity. Yet there is no study which reports the direct neural recording and its pattern during in-vivo progression of the cancer. We performed daily neural recordings from male and female mice bearing orthotopic metastasizing- melanomas and melanomas with low metastatic poential, derived from B16-F10 and B16-F1 cells, respectively. Further, to explore the origins of neural activity, 6-Hydroxidopamine mediated chemical sympathectomy was performed followed by daily microneurographic recordings. We also performed the daily bioluminescent imaging to track in vivo growth of primary tumors and distant metastasis to the cranial area. Our results show that metastasizing tumors display high levels of neural activity while tumors with low metastatic potential lack it indicating that the presence of neural activity is linked to the metastasizing potential of the tumors. Moreover, the neural activity is not continuous over the tumor progression and has a sex-specific temporal patterns where males have two peaks of high neural activity while females show a single peak. The neural peak activity originated in peripheral sympathetic nerves as sympathectomy completely eliminated the peak activity in both sexes. Peak activities were highly correlated with the distant metastasis in both sexes. These results show that sympathetic neural activity is crucially involved in tumor metastasis and has sex-specific role in malignancy initiation.

Spatial and amplitude dynamics of neurostimulation: Insights from the acute intrahippocampal kainate seizure mouse model.

OBJECTIVE: Neurostimulation is an emerging treatment for patients with drug-resistant epilepsy, which is used to suppress, prevent, and terminate seizure activity. Unfortunately, after implantation and despite best clinical practice, most patients continue to have persistent seizures even after years of empirical optimization. The objective of this study is to determine optimal spatial and amplitude properties of neurostimulation in inhibiting epileptiform activity in an acute hippocampal seizure model. METHODS: We performed high-throughput testing of high-frequency focal brain stimulation in the acute intrahippocampal kainic acid mouse model of status epilepticus. We evaluated combinations of six anatomic targets and three stimulus amplitudes. RESULTS: We found that the spike-suppressive effects of high-frequency neurostimulation are highly dependent on the stimulation amplitude and location, with higher amplitude stimulation being significantly more effective. Epileptiform spiking activity was significantly reduced with ipsilateral 250 µA stimulation of the CA1 and CA3 hippocampal regions with 21.5% and 22.2% reductions, respectively. In contrast, we found that spiking frequency and amplitude significantly increased with stimulation of the ventral hippocampal commissure. We further found spatial differences with broader effects from CA1 versus CA3 stimulation. SIGNIFICANCE: These findings demonstrate that the effects of therapeutic neurostimulation in an acute hippocampal seizure model are highly dependent on the location of stimulation and stimulus amplitude. We provide a platform to optimize the anti-seizure effects of neurostimulation, and demonstrate that an exploration of the large electrical parameter and location space can improve current modalities for treating epilepsy. PLAIN LANGUAGE SUMMARY: In this study, we tested how electrical pulses in the brain can help control seizures in mice. We found that the electrode's placement and the stimulation amplitude had a large effect on outcomes. Some brain regions, notably nearby CA1 and CA3, responded positively with reduced seizure-like activities, while others showed increased activity. These findings emphasize that choosing the right spot for the electrode and adjusting the strength of electrical pulses are both crucial when considering neurostimulation treatments for epilepsy.

Selective chronic recording in small nerve fascicles of sciatic nerve with carbon nanotube yarns in rats.

Objective. The primary challenge faced in the field of neural rehabilitation engineering is the limited advancement in nerve interface technology, which currently fails to match the mechanical properties of small-diameter nerve fascicles. Novel developments are necessary to enable long-term, chronic recording from a multitude of small fascicles, allowing for the recovery of motor intent and sensory signals.Approach. In this study, we analyze the chronic recording capabilities of carbon nanotube yarn electrodes in the peripheral somatic nervous system. The electrodes were surgically implanted in the sciatic nerve's three individual fascicles in rats, enabling the recording of neural activity during gait. Signal-to-noise ratio (SNR) and information theory were employed to analyze the data, demonstrating the superior recording capabilities of the electrodes. Flat interface nerve electrode and thin-film longitudinal intrafascicular electrode electrodes were used as a references to assess the results from SNR and information theory analysis.Main results. The electrodes exhibited the ability to record chronic signals with SNRs reaching as high as 15 dB, providing 12 bits of information for the sciatic nerve, a significant improvement over previous methods. Furthermore, the study revealed that the SNR and information content of the neural signals remained consistent over a period of 12 weeks across three different fascicles, indicating the stability of the interface. The signals recorded from these electrodes were also analyzed for selectivity using information theory metrics, which showed an information sharing of approximately 1.4 bits across the fascicles.Significance. The ability to safely and reliably record from multiple fascicles of different nerves simultaneously over extended periods of time holds substantial implications for the field of neural and rehabilitation engineering. This advancement addresses the limitation of current nerve interface technologies and opens up new possibilities for enhancing neural rehabilitation and control.

Mechanisms of Neurostimulation for Epilepsy.

This review discusses the use of neurostimulation therapies for epilepsy treatment, including vagal nerve stimulation, responsive neurostimulation, and deep brain stimulation. Different therapeutic strategies and their underlying mechanisms are explored, with a focus on optimizing parameters for seizure reduction. The review also highlights the paradigm shift toward a more diverse and multimodal approach to deep brain neuromodulation.

Spatial and Amplitude Dynamics of Neurostimulation: Insights from the Acute Intrahippocampal Kainate Seizure Mouse Model.

Objective: Neurostimulation is an emerging treatment for patients with medically refractory epilepsy, which is used to suppress, prevent, and terminate seizure activity. Unfortunately, after implantation and despite best clinical practice, most patients continue to have persistent seizures even after years of empirical optimization. The objective of this study is to determine optimal spatial and amplitude properties of neurostimulation in inhibiting epileptiform activity in an acute hippocampal seizure model. Methods: We performed high-throughput testing of high-frequency focal brain stimulation in the acute intrahippocampal kainic acid mouse model of temporal lobe epilepsy. We evaluated combinations of six anatomic targets and three stimulus amplitudes. Results: We found that the spike-suppressive effects of high-frequency neurostimulation are highly dependent on the stimulation amplitude and location, with higher amplitude stimulation being significantly more effective. Epileptiform spiking activity was significantly reduced with ipsilateral 250 µA stimulation of the CA1 and CA3 hippocampal regions with 21.5% and 22.2% reductions, respectively. In contrast, we found that spiking frequency and amplitude significantly increased with stimulation of the ventral hippocampal commissure. We further found spatial differences with broader effects from CA1 versus CA3 stimulation. Significance: These findings demonstrate that the effects of therapeutic neurostimulation in an acute hippocampal seizure model are highly dependent on the location of stimulation and stimulus amplitude. We provide a platform to optimize the anti-seizure effects of neurostimulation, and demonstrate that an exploration of the large electrical parameter and location space can improve current modalities for treating epilepsy. Key Points: Evaluated spatial and temporal parameters of neurostimulation in a mouse model of acute seizuresBrief bursts of high-frequency (100 Hz) stimulation effectively interrupted epileptiform activity.The suppressive effect was highly dependent on stimulation amplitude and was maximal at the ipsilateral CA1 and CA3 regions.Pro-excitatory effects were identified with high-amplitude high-frequency stimulation at the ventral hippocampal commissure and contralateral CA1.

Theta waves, neural spikes and seizures can propagate by ephaptic coupling in vivo.

Electric field coupling has been shown to be responsible for non-synaptic neural activity propagation in hippocampal slices and cortical slices. Epileptiform and slow-wave sleep activity can propagate by electric field coupling without using synaptic connections at speeds of ~0.1 m/s in vitro. However, the characteristics of the events that can propagate using electric field coupling through a volume conductor in vivo have not been studied. Thus, we tested the hypothesis that various types of neural signals such as interictal spikes, theta waves and seizures could propagate in vivo across a transection in the hippocampus. We induced epileptiform activity in 4 rats under anesthesia by injecting 4-aminopyridine in the temporal region of the hippocampus, four recording electrodes were inserted along the longitudinal axis of the hippocampus. A transection was made between the electrodes to study the propagation of the neural activity. Although 54% of the interictal spikes could propagate through the cut, only those spikes with a high amplitude and short duration had a high probability to do so. 70% of seizure events could propagate through the cut but parameters distinguishing between propagating and non-propagating seizure events could not be identified. Theta activity was also observed to propagate at a mean speed of 0.16 ± 0.12 m/s in the characteristic range of propagation using electric field coupling through the transection. The electric field volume conduction mechanism was confirmed by showing that propagation was blocked by placing a dielectric layer within the cut. The speed of propagation was not affected by the transection thereby providing further evidence that various types of neural signals including activity in the theta range can propagate by electric field coupling in-vivo.

Pulse-frequency-dependent resonance in a population of pyramidal neuron models.

Stochastic resonance is known as a phenomenon whereby information transmission of weak signal or subthreshold stimuli can be enhanced by additive random noise with a suitable intensity. Another phenomenon induced by applying deterministic pulsatile electric stimuli with a pulse frequency, commonly used for deep brain stimulation (DBS), was also shown to improve signal-to-noise ratio in neuron models. The objective of this study was to test the hypothesis that pulsatile high-frequency stimulation could improve the detection of both sub- and suprathreshold synaptic stimuli by tuning the frequency of the stimulation in a population of pyramidal neuron models. Computer simulations showed that mutual information estimated from a population of neural spike trains displayed a typical resonance curve with a peak value of the pulse frequency at 80-120 Hz, similar to those utilized for DBS in clinical situations. It is concluded that a "pulse-frequency-dependent resonance" (PFDR) can enhance information transmission over a broad range of synaptically connected networks. Since the resonance frequency matches that used clinically, PFDR could contribute to the mechanism of the therapeutic effect of DBS.

Low-frequency stimulation of a fiber tract in bilateral temporal lobe epilepsy.

OBJECTIVE: Pharmacoresistant bilateral mesial temporal lobe epilepsy often implies poor resective surgical candidacy. Low-frequency stimulation of a fiber tract connected to bilateral hippocampi, the fornicodorsocommissural tract, has been shown to be safe and efficacious in reducing seizures in a previous short-term study. Here, we report a single-blinded, within-subject control, long-term deep-brain stimulation trial of low-frequency stimulation of the fornicodorsocommissural tract in bilateral mesial temporal lobe epilepsy. Outcomes of interest included safety with respect to verbal memory scores and reduction of seizure frequency. METHODS: Our enrollment goal was 16 adult subjects to be randomized to 2-Hz or 5-Hz low-frequency stimulation of the fornicodorsocommissural tract starting at 2 mA. The study design consisted of four two-month blocks of stimulation with a 50%-duty cycle, alternating with two-month blocks of no stimulation. RESULTS: We terminated the study after enrollment of five subjects due to slow accrual. Fornicodorsocommissural tract stimulation elicited bilateral hippocampal evoked responses in all subjects. Three subjects underwent implantation of pulse generators and long-term low-frequency stimulation with mean monthly seizures of 3.14 ±â€¯2.67 (median 3.0 [IQR 1-4.0]) during stimulation-off blocks, compared with 0.96 ±â€¯1.23 (median 1.0 [IQR 0-1.0]) during stimulation-on blocks (p = 0.0005) during the blinded phase. Generalized Estimating Equations showed that low-frequency stimulation reduced monthly seizure-frequency by 0.71 per mA (p < 0.001). Verbal memory scores were stable with no psychiatric complications or other adverse events. SIGNIFICANCE: The results demonstrate feasibility of stimulating both hippocampi using a single deep-brain stimulation electrode in the fornicodorsocommissural tract, efficacy of low-frequency stimulation in reducing seizures, and safety as regards verbal memory.

Decoding Vagus-Nerve Activity with Carbon Nanotube Sensors in Freely Moving Rodents.

The vagus nerve is the largest autonomic nerve and a major target of stimulation therapies for a wide variety of chronic diseases. However, chronic recording from the vagus nerve has been limited, leading to significant gaps in our understanding of vagus nerve function and therapeutic mechanisms. In this study, we use a carbon nanotube yarn (CNTY) biosensor to chronically record from the vagus nerves of freely moving rats for over 40 continuous hours. Vagal activity was analyzed using a variety of techniques, such as spike sorting, spike-firing rates, and interspike intervals. Many spike-cluster-firing rates were found to correlate with food intake, and the neural-firing rates were used to classify eating and other behaviors. To our knowledge, this is the first chronic recording and decoding of activity in the vagus nerve of freely moving animals enabled by the axon-like properties of the CNTY biosensor in both size and flexibility and provides an important step forward in our ability to understand spontaneous vagus-nerve function.

Subthreshold Oscillating Waves in Neural Tissue Propagate by Volume Conduction and Generate Interference.

Subthreshold neural oscillations have been observed in several brain regions and can influence the timing of neural spikes. However, the spatial extent and function of these spontaneous oscillations remain unclear. To study the mechanisms underlying these oscillations, we use optogenetic stimulation to generate oscillating waves in the longitudinal hippocampal slice expressing optopatch proteins. We found that optogenetic stimulation can generate two types of neural activity: suprathreshold neural spikes and subthreshold oscillating waves. Both waves could propagate bidirectionally at similar speeds and go through a transection of the tissue. The propagating speed is independent of the oscillating frequency but increases with increasing amplitudes of the waves. The endogenous electric fields generated by oscillating waves are about 0.6 mV/mm along the dendrites and about 0.3 mV/mm along the cell layer. We also observed that these oscillating waves could interfere with each other. Optical stimulation applied simultaneously at each slice end generated a larger wave in the middle of the tissue (constructive interference) or destructive interference with laser signals in opposite phase. However, the suprathreshold neural spikes were annihilated when they collided. Finally, the waves were not affected by the NMDA blocker (APV) and still propagated in the presence of tetrodotoxin (TTX) but at a significantly lower amplitude. The role of these subthreshold waves in neural function is unknown, but the results show that at low amplitude, the subthreshold propagating waves lack a refractory period allowing a novel analog form of preprocessing of neural activity by interference independent of synaptic transmission.

Delta oscillation underlies the interictal spike changes after repeated transcranial direct current stimulation in a rat model of chronic seizures.

BACKGROUND: Transcranial direct current stimulation (tDCS) provides a noninvasive polarity-specific constant current to treat epilepsy, through a mechanism possibly involving excitability modulation and neural oscillation. OBJECTIVE: To determine whether EEG oscillations underlie the interictal spike changes after tDCS in rats with chronic spontaneous seizures. METHODS: Rats with kainic acid-induced spontaneous seizures were subjected to cathodal tDCS or sham stimulation for 5 consecutive days. Video-EEG recordings were collected immediately pre- and post-stimulation and for the subsequent 2 weeks following stimulation. The acute pre-post stimulation and subacute follow-up changes of interictal spikes and EEG oscillations in tDCS-treated rats were compared with sham. Ictal EEG with seizure behaviors, hippocampal brain-derived neurotrophic factor (BDNF) protein expression, and mossy fiber sprouting were compared between tDCS and sham rats. RESULTS: Interictal spike counts were reduced immediately following tDCS with augmented delta and diminished beta and gamma oscillations compared with sham. Cathodal tDCS also enhanced delta oscillations in normal rats. However, increased numbers of interictal spikes with a decrease of delta and theta oscillations were observed in tDCS-treated rats compared with sham during the following 2 weeks after stimulation. Resuming tDCS suppressed the increase of interictal spike activity. In tDCS rats, hippocampal BDNF protein expression was decreased while mossy fiber sprouting did not change compared with sham. CONCLUSIONS: The inverse relationship between the changes of delta oscillation and interictal spikes during tDCS on and off stimulation periods indicates that an enhanced endogenous delta oscillation underlies the tDCS inhibitory effect on epileptic excitability.

Neural recruitment by ephaptic coupling in epilepsy.

OBJECTIVE: One of the challenges in treating patients with drug-resistant epilepsy is that the mechanisms of seizures are unknown. Most current interventions are based on the assumption that epileptic activity recruits neurons and progresses by synaptic transmission. However, several experimental studies have shown that neural activity in rodent hippocampi can propagate independently of synaptic transmission. Recent studies suggest these waves are self-propagating by electric field (ephaptic) coupling. In this study, we tested the hypothesis that neural recruitment during seizures can occur by electric field coupling. METHODS: 4-Aminopyridine was used in both in vivo and in vitro preparation to trigger seizures or epileptiform activity. A transection was made in the in vivo hippocampus and in vitro hippocampal and cortical slices to study whether the induced seizure activity can recruit neurons across the gap. A computational model was built to test whether ephaptic coupling alone can account for neural recruitment across the transection. The model prediction was further validated by in vitro experiments. RESULTS: Experimental results show that electric fields generated by seizure-like activity in the hippocampus both in vitro and in vivo can recruit neurons locally and through a transection of the tissue. The computational model suggests that the neural recruitment across the transection is mediated by electric field coupling. With in vitro experiments, we show that a dielectric material can block the recruitment of epileptiform activity across a transection, and that the electric fields measured within the gap are similar to those predicted by model simulations. Furthermore, this nonsynaptic neural recruitment is also observed in cortical slices, suggesting that this effect is robust in brain tissue. SIGNIFICANCE: These results indicate that ephaptic coupling, a nonsynaptic mechanism, can underlie neural recruitment by a small electric field generated by seizure activity and could explain the low success rate of surgical transections in epilepsy patients.

Model-based analysis of implanted hypoglossal nerve stimulation for the treatment of obstructive sleep apnea.

STUDY OBJECTIVES: Individuals with obstructive sleep apnea (OSA), characterized by frequent sleep disruptions from tongue muscle relaxation and airway blockage, are known to benefit from on-demand electrical stimulation of the hypoglossal nerve. Hypoglossal nerve stimulation (HNS) therapy, which activates the protrusor muscles of the tongue during inspiration, has been established in multiple clinical studies as safe and effective, but the mechanistic understanding for why some stimulation parameters work better than others has not been thoroughly investigated. METHODS: In this study, we developed a detailed biophysical model that can predict the spatial recruitment of hypoglossal nerve fascicles and axons within these fascicles during stimulation through nerve cuff electrodes. Using this model, three HNS programming scenarios were investigated including grouped cathode (---), single cathode (o-o), and guarded cathode bipolar (+-+) electrode configurations. RESULTS: Regardless of electrode configuration, nearly all hypoglossal nerve axons circumscribed by the nerve cuff were recruited for stimulation amplitudes <3 V. Within this range, monopolar configurations required lower stimulation amplitudes than the guarded bipolar configuration to elicit action potentials within hypoglossal nerve axons. Further, the spatial distribution of the activated axons was more uniform for monopolar versus guarded bipolar configurations. CONCLUSIONS: The computational models predicted that monopolar HNS provided the lowest threshold and the least sensitivity to rotational angle of the nerve cuff around the hypoglossal nerve; however, this setting also increased the likelihood for current leakage outside the nerve cuff, which could potentially activate axons in unintended branches of the hypoglossal nerve. CLINICAL TRIAL REGISTRATION: NCT01161420.

Direct measurement of vagal tone in rats does not show correlation to HRV.

The vagus nerve is the largest autonomic nerve, innervating nearly every organ in the body. "Vagal tone" is a clinical measure believed to indicate overall levels of vagal activity, but is measured indirectly through the heart rate variability (HRV). Abnormal HRV has been associated with many severe conditions such as diabetes, heart failure, and hypertension. However, vagal tone has never been directly measured, leading to disagreements in its interpretation and influencing the effectiveness of vagal therapies. Using custom carbon nanotube yarn electrodes, we were able to chronically record neural activity from the left cervical vagus in both anesthetized and non-anesthetized rats. Here we show that tonic vagal activity does not correlate with common HRV metrics with or without anesthesia. Although we found that average vagal activity is increased during inspiration compared to expiration, this respiratory-linked signal was not correlated with HRV either. These results represent a clear advance in neural recording technology but also point to the need for a re-interpretation of the link between HRV and "vagal tone".

Chronic neural activity recorded within breast tumors.

Nerve fibers are known to reside within malignant tumors and the greater the neuronal density the worse prognosis for the patient. Recent discoveries using tumor bearing animal models have eluded to the autonomic nervous system having a direct effect on tumor growth and metastasis. We report the first direct and chronic in vivo measurements of neural activity within tumors. Using a triple-negative mammary cancer mouse model and chronic neural interface techniques, we have recorded neural activity directly within the tumor mass while the tumor grows and metastasizes. The results indicate that there is a strong connection between the autonomic nervous system and the tumor and could help uncover the mechanisms of tumor growth and metastasis.

Transcranial direct current stimulation alleviates seizure severity in kainic acid-induced status epilepticus rats.

Status epilepticus (SE) is a state of prolonged and repeated seizures that can lead to permanent brain damage or life-threatening conditions. Transcranial direct current stimulation (tDCS) non-invasively provides a polarity-specific electric current to modulate brain excitability. Little is known about the therapeutic potential of tDCS in SE. Here, we aim to determine the tDCS effects on seizure severity, EEG and post-SE consequences in rats with kainic acid (KA)-induced SE. Rats were subjected to cathodal tDCS or sham stimulation over the dorsal hippocampus for 5 days. KA was intraperitoneally injected to induce SE. We used continuous video-EEG recording to monitor seizure activity, immunostaining and Timm staining to evaluate neuron counts and mossy fiber sprouting, and ELISA for Brain-derived neurotrophic factor (BDNF) protein measurement. Two featured EEG patterns, gamma ranged high-frequency polyspikes and low-frequency spike-and-wave complexes, were identified in the hippocampal CA1 of KA-induced SE rats. tDCS elicited a significant decrease in severe seizures of Racine stages 4-5 in KA-induced SE rats. tDCS-treated rats manifested diminished high-frequency oscillation during SE, decreased chronic spontaneous spike activities and mossy fiber sproutings compared to sham. tDCS-treated rats also exhibited significantly lower hippocampal BDNF protein levels than sham immediately and 4 weeks after SE. A positive correlation between the hippocampal BDNF level and the seizure severity of SE was found. Altogether, our results show that repeated cathodal tDCS can mitigate seizure severity, alter ictal EEG pattern and reduce the chronic adverse consequences in KA-induced SE rats, supporting the therapeutic potential of tDCS in severe prolonged epileptic seizures.

Comparison of fiber tract low frequency stimulation to focal and ANT stimulation in an acute rat model of focal cortical seizures.

BACKGROUND: Current implementations of direct brain stimulation for epilepsy in patients involve high-frequency (HFS) electrical current and targeting of grey matter. Studies have shown that low-frequency (LFS) fiber-tract stimulation may also prove effective. To compare the efficacy of high-frequency grey matter stimulation to the low-frequency fiber tract stimulation technique a well-controlled set of experiments using a single animal model of epilepsy is needed. OBJECTIVE: The goal of this study was to determine the relative efficacy of different direct brain stimulation techniques for suppressing seizures using an acute rat model of focal cortical seizures. METHODS: 4-AP was injected into the S1 region of cortex in rodents over 3 h. LFPs were recorded from the seizure focus and mirror focus to monitor seizure frequency during the experiments. CC-LFS, HFS-ANT, Focal-HFS, or a transection of the CC was applied. RESULTS: Stimulation of the CC yielded a 65% ±14% (p = 0.0014) reduction of seizures in the focus and a 97% ±15% (p = 0.0026) reduction in the mirror focus (n = 7). By comparison transection of the CC produced a 65% ±18% reduction in the focus and a non-statistically significant reduction of 57% ±18% (p = 0.1381) in the mirror focus (n = 5). All other methods of stimulation failed to have a statistically significant effect on seizure suppression. CONCLUSIONS: LFS of the CC is the only method of stimulation to significantly reduce seizure frequency in this model of focal cortical seizures. These results support the hypothesis that LFSof fiber tracts has significant potential for seizure control.

Sonic Hedgehog is expressed by hilar mossy cells and regulates cellular survival and neurogenesis in the adult hippocampus.

Sonic hedgehog (Shh) is a multifunctional signaling protein governing pattern formation, proliferation and cell survival during embryogenesis. In the adult brain, Shh has neurotrophic function and is implicated in hippocampal neurogenesis but the cellular source of Shh in the hippocampus remains ill defined. Here, we utilize a gene expression tracer allele of Shh (Shh-nlacZ) which allowed the identification of a subpopulation of hilar neurons known as mossy cells (MCs) as a prominent and dynamic source of Shh within the dentate gyrus. AAV-Cre mediated ablation of Shh in the adult dentate gyrus led to a marked degeneration of MCs. Conversely, chemical stimulation of hippocampal neurons using the epileptogenic agent kainic acid (KA) increased the number of Shh+ MCs indicating that the expression of Shh by MCs confers a survival advantage during the response to excitotoxic insults. In addition, ablation of Shh in the adult dentate gyrus led to increased neural precursor cell proliferation and their migration into the subgranular cell layer demonstrating that MCs-generated Shh is a key modulator of hippocampal neurogenesis.