Further studies in rat models of neural disinhibition: behavioural, in vivo electrophysiological, and translational imaging studies

Neural disinhibition, i.e., reduced inhibitory GABA transmission, has been implicated in various neuropsychiatric disorders. In particular, GABAergic abnormalities in the hippocampus and the striatum have been associated with schizophrenia and Tourette Syndrome, respectively. Regional neural disinhibition may affect behaviour by disrupting regional neural function and by causing changes in projection sites of the disinhibited region. In schizophrenia, one of such projection sites is the septum and in Tourette Syndrome the motor cortex. To further examine the impact of regional disinhibition (ventral hippocampus or anterior dorsal striatum), we combined intra-cerebral microinfusions of a GABA-A receptor antagonist (picrotoxin) with translational neural imaging (MRS, rsFC MRI), electrophysiological measurements and behavioural methods (locomotor activity and prepulse inhibition) in Lister hooded rats. First, we used 1H-Magnetic Resonance Spectroscopy (MRS) at 7T, to measure neuro-metabolites in the septum following ventral hippocampal disinhibition (Chapter 2). This study was based on our previous Singlephoton emission computed tomography (SPECT) imaging study that revealed marked metabolic activation in several extra-hippocampal sites, including the septum, after ventral hippocampus disinhibition (Williams et al., in preparation). This experiment demonstrated no clear effects of ventral hippocampal disinhibition on any of the neuro-metabolites measured in the septum, including glutamate, glutamine and GABA, indicating that marked acute metabolic activation in the lateral septum (as detected by SPECT) is potentially not accompanied by neuro-metabolites changes measurable by MRS (possibly reflecting homeostatic metabolic mechanisms). Second, we used electrophysiological measurements in anaesthetised rats (Chapter 3) to examine the effects of striatal disinhibition, induced by picrotoxin infusions (300 ng picrotoxin in 0.5 µl saline) into the anterior dorsal striatum, on neural activity in the vicinity of the infusion site. Our findings revealed large local field potential (LFP) spike-wave discharges, consisting of a single negative spike followed by a positive wave. Furthermore, picrotoxin in the dorsal striatum enhanced multi-unit burst firing, reflected by significant increases in mean spike frequency in burst per block, mean peak spike frequency in burst and percentage spikes in burst and by increased frequency of bursts, reflected by significant decreases of mean interburst interval per block. This is a new finding in the striatum and is consistent with disinhibition-induced enhancement of burst firing that we previously reported in the prefrontal cortex and hippocampus (Pezze et al., 2014; McGarrity et al., 2017), suggesting that in all these regions, GABA-A receptor-mediated inhibition is particularly important to control neural burst firing. No tic-like movements were visible in the anesthetised rats. Third, we characterised tic-like forelimb movements caused by infusing picrotoxin into the right anterior dorsal striatum (300 ng and 200 ng picrotoxin in 0.5 µl saline) of rats (Chapter 4). Such infusions reliably induced tic-like movements in the left forelimb within the same rat and across rats. Most rats expressed highest frequency of tic-like movements in the first 5 to 35 min post-infusion. Tic-like movements were characterised. The most common tic-like movement involved the rat to lift its left forelimb, thereby rotating its head and torso to the right around the body’s long axis, before putting the left forelimb back down again, and thereby moving its head and torso into its starting position. There were also some more pronounced forelimb movements that lasted for several seconds and led to a whole rotation of the body around its long axis. The movements observed in this study were compared to tics in Tourette syndrome. Fourth, we investigated the impact of anterior dorsal striatal disinhibition (300 ng picrotoxin in 0.5 µl saline) on prepulse inhibition (PPI) of the acoustic startle response and locomotor activity (Chapter 5). Picrotoxin infusion caused tic-like movements, had no effect on prepulse inhibition, tended to reduce startle and significantly increased locomotor activity and fine motor count. No PPI deficit following striatal disinhibition indicates that GABAergic inhibition in the dorsal striatum is not critical for prepulse inhibition. Our finding that marked tic-like movements were produced alongside intact PPI does not support the possibility that PPI disruption is necessary for tic-like movements (as suggested by Swerdlow and colleagues). The timeline of the fine motor count was similar to the timeline of the tic-like movements, and therefore, fine motor counts may thus be used as an automated measure of tic-like movements. The locomotor hyperactivity alongside tic-like movements indicates that dorsal striatal activity is not only involved in generating movements of individual body parts, but also in modulating locomotor activity and suggests that striatal disinhibition that contributes to tic-like movements may also contribute to hyperactivity, which is often comorbid with Tourette Syndrome (Robertson, 2015). Lastly, we aimed to provide proof of principle that standard resting state functional connectivity Magnetic Resonance Imaging (rsFC MRI) measurements are possible in rats with pre-implanted guide cannulae and microinfusions in the anterior dorsal striatum (Chapter 6). Although we obtained sufficient quality rsFC MRI data to reveal the resting-state default mode network of an unoperated rat using the anterior cingulate as a seed, we were unable to obtain any resting-state default mode network of an operated rat, due to the significant signal loss from the guide cannula. We also tried to obtain spectra from the motor cortex following striatal disinhibition (using MRS), however, were unable to get good Signal to Noise ratio values for the water signal of the voxel placed in the motor cortex (SNR H2O). Although some neuro-metabolites, including glutamate, were measurable in the motor cortex of rats with a dorsal striatal guide cannula, others, including GABA, were not. When comparing the SNR H2O values between our studies, it appears that SNR H2O values decrease with increased distance between regions (disinhibited region and ROI for spectrum). Concluding, disinhibiting the ventral hippocampus, has no clear effects on any neuro-metabolites in the septum, as measured by MRS. Dorsal striatal disinhibition causes large spike wave discharges and enhances burst firing in the striatum under anaesthesia. In behaving rats, striatal disinhibition causes tic-like movements, increases locomotor activity and fine movement counts, and has no effect on prepulse inhibition. Obtaining sufficient quality rsFC MRI data to reveal the resting-state default mode network is not possible following implantation of a guide cannula, as the cannula leads to too much signal loss. The signal for MRS is too poor with striatal cannula implants and a voxel placed in the motor cortex

Further studies in rat models of neural disinhibition: behavioural, in vivo electrophysiological, and translational imaging studies

Neural disinhibition, i.e., reduced inhibitory GABA transmission, has been implicated in various neuropsychiatric disorders. In particular, GABAergic abnormalities in the hippocampus and the striatum have been associated with schizophrenia and Tourette Syndrome, respectively. Regional neural disinhibition may affect behaviour by disrupting regional neural function and by causing changes in projection sites of the disinhibited region. In schizophrenia, one of such projection sites is the septum and in Tourette Syndrome the motor cortex. To further examine the impact of regional disinhibition (ventral hippocampus or anterior dorsal striatum), we combined intra-cerebral microinfusions of a GABA-A receptor antagonist (picrotoxin) with translational neural imaging (MRS, rsFC MRI), electrophysiological measurements and behavioural methods (locomotor activity and prepulse inhibition) in Lister hooded rats. First, we used 1H-Magnetic Resonance Spectroscopy (MRS) at 7T, to measure neuro-metabolites in the septum following ventral hippocampal disinhibition (Chapter 2). This study was based on our previous Singlephoton emission computed tomography (SPECT) imaging study that revealed marked metabolic activation in several extra-hippocampal sites, including the septum, after ventral hippocampus disinhibition (Williams et al., in preparation). This experiment demonstrated no clear effects of ventral hippocampal disinhibition on any of the neuro-metabolites measured in the septum, including glutamate, glutamine and GABA, indicating that marked acute metabolic activation in the lateral septum (as detected by SPECT) is potentially not accompanied by neuro-metabolites changes measurable by MRS (possibly reflecting homeostatic metabolic mechanisms). Second, we used electrophysiological measurements in anaesthetised rats (Chapter 3) to examine the effects of striatal disinhibition, induced by picrotoxin infusions (300 ng picrotoxin in 0.5 µl saline) into the anterior dorsal striatum, on neural activity in the vicinity of the infusion site. Our findings revealed large local field potential (LFP) spike-wave discharges, consisting of a single negative spike followed by a positive wave. Furthermore, picrotoxin in the dorsal striatum enhanced multi-unit burst firing, reflected by significant increases in mean spike frequency in burst per block, mean peak spike frequency in burst and percentage spikes in burst and by increased frequency of bursts, reflected by significant decreases of mean interburst interval per block. This is a new finding in the striatum and is consistent with disinhibition-induced enhancement of burst firing that we previously reported in the prefrontal cortex and hippocampus (Pezze et al., 2014; McGarrity et al., 2017), suggesting that in all these regions, GABA-A receptor-mediated inhibition is particularly important to control neural burst firing. No tic-like movements were visible in the anesthetised rats. Third, we characterised tic-like forelimb movements caused by infusing picrotoxin into the right anterior dorsal striatum (300 ng and 200 ng picrotoxin in 0.5 µl saline) of rats (Chapter 4). Such infusions reliably induced tic-like movements in the left forelimb within the same rat and across rats. Most rats expressed highest frequency of tic-like movements in the first 5 to 35 min post-infusion. Tic-like movements were characterised. The most common tic-like movement involved the rat to lift its left forelimb, thereby rotating its head and torso to the right around the body’s long axis, before putting the left forelimb back down again, and thereby moving its head and torso into its starting position. There were also some more pronounced forelimb movements that lasted for several seconds and led to a whole rotation of the body around its long axis. The movements observed in this study were compared to tics in Tourette syndrome. Fourth, we investigated the impact of anterior dorsal striatal disinhibition (300 ng picrotoxin in 0.5 µl saline) on prepulse inhibition (PPI) of the acoustic startle response and locomotor activity (Chapter 5). Picrotoxin infusion caused tic-like movements, had no effect on prepulse inhibition, tended to reduce startle and significantly increased locomotor activity and fine motor count. No PPI deficit following striatal disinhibition indicates that GABAergic inhibition in the dorsal striatum is not critical for prepulse inhibition. Our finding that marked tic-like movements were produced alongside intact PPI does not support the possibility that PPI disruption is necessary for tic-like movements (as suggested by Swerdlow and colleagues). The timeline of the fine motor count was similar to the timeline of the tic-like movements, and therefore, fine motor counts may thus be used as an automated measure of tic-like movements. The locomotor hyperactivity alongside tic-like movements indicates that dorsal striatal activity is not only involved in generating movements of individual body parts, but also in modulating locomotor activity and suggests that striatal disinhibition that contributes to tic-like movements may also contribute to hyperactivity, which is often comorbid with Tourette Syndrome (Robertson, 2015). Lastly, we aimed to provide proof of principle that standard resting state functional connectivity Magnetic Resonance Imaging (rsFC MRI) measurements are possible in rats with pre-implanted guide cannulae and microinfusions in the anterior dorsal striatum (Chapter 6). Although we obtained sufficient quality rsFC MRI data to reveal the resting-state default mode network of an unoperated rat using the anterior cingulate as a seed, we were unable to obtain any resting-state default mode network of an operated rat, due to the significant signal loss from the guide cannula. We also tried to obtain spectra from the motor cortex following striatal disinhibition (using MRS), however, were unable to get good Signal to Noise ratio values for the water signal of the voxel placed in the motor cortex (SNR H2O). Although some neuro-metabolites, including glutamate, were measurable in the motor cortex of rats with a dorsal striatal guide cannula, others, including GABA, were not. When comparing the SNR H2O values between our studies, it appears that SNR H2O values decrease with increased distance between regions (disinhibited region and ROI for spectrum). Concluding, disinhibiting the ventral hippocampus, has no clear effects on any neuro-metabolites in the septum, as measured by MRS. Dorsal striatal disinhibition causes large spike wave discharges and enhances burst firing in the striatum under anaesthesia. In behaving rats, striatal disinhibition causes tic-like movements, increases locomotor activity and fine movement counts, and has no effect on prepulse inhibition. Obtaining sufficient quality rsFC MRI data to reveal the resting-state default mode network is not possible following implantation of a guide cannula, as the cannula leads to too much signal loss. The signal for MRS is too poor with striatal cannula implants and a voxel placed in the motor cortex

The role of the insula in the generation of motor tics and the experience of the premonitory urge-to-tic in Tourette syndrome

Tourette syndrome (TS) is a neurological disorder of childhood onset that is characterised by the occurrence of motor and vocal tics. TS is associated with cortical-striatal-thalamic-cortical circuit [CSTC] dysfunction and hyper-excitability of cortical limbic and motor regions that are thought to lead to the occurrence of tics. Importantly, individuals with TS often report that their tics are preceded by ‘premonitory sensory/urge phenomena’ (PU) that are described as uncomfortable bodily sensations that precede the execution of a tic and are experienced as a strong urge for motor discharge. While the precise role played by PU in the occurrence of tics is largely unknown, they are nonetheless of considerable theoretical and clinical importance, not least because they form the core component in many behavioural therapies used in the treatment of tic disorders. Several lines of evidence indicate that the insular cortex may play a particularly important role in the generation of PU in TS and ‘urges-for-action’ more generally. In the current study we utilised voxel-based morphometry techniques together with ‘seed-to-voxel’ structural covariance network (SCN) mapping to investigate the putative role played by the right insular cortex in the generation of motor tics and the experience of PU in a relatively large group of young people TS. We demonstrate that clinical measures of motor tic severity and PU are uncorrelated with one another, that motor tic severity and PU scores are associated with separate regions of the insular cortex, and that the insula is associated with different structural covariance networks in individuals with TS compared to a matched group of typically developing individuals

Hippocampal Disinhibition Reduces Contextual and Elemental Fear Conditioning While Sparing the Acquisition of Latent Inhibition

Hippocampal neural disinhibition, i.e., reduced GABAergic inhibition, is a key feature of schizophrenia pathophysiology. The hippocampus is an important part of the neural circuitry that controls fear conditioning and can also modulate prefrontal and striatal mechanisms, including dopamine signaling, which play a role in salience modulation. Consequently, hippocampal neural disinhibition may contribute to impairments in fear conditioning and salience modulation reported in schizophrenia. Therefore, we examined the effect of ventral hippocampus (VH) disinhibition in male rats on fear conditioning and salience modulation, as reflected by latent inhibition (LI), in a conditioned emotional response (CER) procedure. A flashing light was used as the conditioned stimulus (CS), and conditioned suppression was used to index conditioned fear. In experiment 1, VH disinhibition via infusion of the GABA-A receptor antagonist picrotoxin before CS pre-exposure and conditioning markedly reduced fear conditioning to both the CS and context; LI was evident in saline-infused controls but could not be detected in picrotoxin-infused rats because of the low level of fear conditioning to the CS. In experiment 2, VH picrotoxin infusions only before CS pre-exposure did not affect the acquisition of fear conditioning or LI. Together, these findings indicate that VH neural disinhibition disrupts contextual and elemental fear conditioning, without affecting the acquisition of LI. The disruption of fear conditioning resembles aversive conditioning deficits reported in schizophrenia and may reflect a disruption of neural processing both within the hippocampus and in projection sites of the hippocampus

Hippocampal disinhibition reduces contextual and elemental fear conditioning while sparing the acquisition of latent inhibition

Hippocampal neural disinhibition, i.e. reduced GABAergic inhibition, is a key feature of schizophrenia pathophysiology. The hippocampus is an important part of the neural circuitry that controls fear conditioning and can also modulate prefrontal and striatal mechanisms, including dopamine signalling, which play a role in salience modulation. Therefore, hippocampal neural disinhibition may contribute to impairments in fear conditioning and salience modulation reported in schizophrenia. To test this hypothesis, we examined the effect of ventral hippocampus (VH) disinhibition in male rats on fear conditioning and salience modulation, as reflected by latent inhibition (LI), in a conditioned emotional response procedure (CER). A flashing light was used as the conditioned stimulus (CS) and conditioned suppression was used to index conditioned fear. In Experiment 1, VH disinhibition via infusion of the GABA-A receptor antagonist picrotoxin prior to CS pre-exposure and conditioning markedly reduced fear conditioning to both the CS and context; LI was evident in saline-infused controls, but could not be detected in picrotoxin-infused rats due to the low level of fear conditioning to the CS. In Experiment 2, VH picrotoxin infusions prior to CS pre-exposure only did not affect the acquisition of fear conditioning or LI. Together, these findings indicate that VH neural disinhibition disrupts contextual and elemental fear conditioning, without affecting the acquisition of LI. The disruption of fear conditioning resembles aversive conditioning deficits reported in schizophrenia and may reflect disruption of neural processing within the hippocampus and its projection sites

Lifetime behavioural changes after exposure to anaesthetics in infant rats

The aim of this study was to assess the effect of acute use of general anaesthetic with or without a surgical procedure, at post-natal day 14 (P14), on behavioural responses in the short-, medium- and long-term, evaluated in open field (OF) and elevated plus-maze (EPM) tests. Fourteen-day-old male Wistar rats were divided into two experimental designs (ED): inhalation and intravenous anaesthetic, and these groups were subdivided into: 1st ED – control (C), isoflurane (ISO), isoflurane/surgery (ISO-SUR); 2nd ED – control (C), fentanyl/S(+)-ketamine (FK) and fentanyl + ketamine-s/surgery (FK-SUR). In the OF the following were found: (a) in the 1st ED: an increase in the locomotor activity in the ISO group at P14, and ISO and ISO-SUR groups at P30; the ISO-SUR group showed a reduced latency to leave the first quadrant at P30 and P60; (b) in the 2nd ED: FK and FK-SUR groups presented increased locomotor activity at P30, and the FK group showed a reduction in the number of faecal boluses. In the EPM the following were found: FK and FK-SUR groups presented an increase in the number of non-protected head-dipping (NPHD) movements and in the number of entries and time spent in open arms at P30; the FK group showed an increased number of protected head-dipping movements, NPHD and entries and time spent in the open arms at P60. The behavioural changes observed may be related to locomotor activity (1st ED) and anxiety level (2nd ED) and they may result from changes in neurotransmitters/hormones (DA, 5HT, CRH) and glutamate/NMDA receptors, respectively