- Top of page
- Materials and Methods
Purpose: Neuropeptide Y (NPY) is an inhibitory neurotransmitter that suppresses focal and generalized seizures in animal models. In this study, we investigated the sites within the thalamocortical circuit that NPY acts to suppress seizures in genetic absence epilepsy rats from Strasbourg (GAERS).
Methods: In conscious freely moving GAERS, NPY was administered via intracerebral microcannulae implanted bilaterally into one of the following regions: primary somatosensory cortex (S1), secondary somatosensory cortex (S2), the primary motor cortex (M1), caudal nucleus reticular thalamus (nRT), or ventrobasal thalamus (VB). Animals received vehicle and up to three doses of NPY, in a randomized order. Electroencephalography (EEG) recordings were carried out for 30 min prior to injection and 90 min after the injection of NPY or vehicle.
Key Findings: Focal microinjections of NPY into the S2 cortex suppressed seizures in a dose-dependent manner, with the response being significantly different at the highest dose (1.5 mm) compared to vehicle for total time in seizures postinjection (7.2 ± 3.0% of saline, p < 0.01) and average number of seizures (9.4 ± 4.9% of saline, p < 0.05). In contrast NPY microinjections into the VB resulted in an aggravation of seizures.
Significance: NPY produces contrasting effects on absence-like seizures in GAERS depending on the site of injection within the thalamocortical circuit. The S2 is the site at which NPY most potently acts to suppress absence-like seizures in GAERS, whereas seizure-aggravating effects are seen in the VB. These results provide further evidence to support the proposition that these electroclinically “generalized” seizures are being driven by a topographically restricted region within the somatosensory cortex.
Neuropeptide Y (NPY) is a 36–amino acid residue member of the pancreatic polypeptide family. Five G protein–coupled NPY receptors have been cloned, which are linked to the inhibition of adenylate cyclase and regulation of intracellular calcium. NPY is colocalized with several other neurotransmitters and is a critical inhibitor of neuronal excitability. Evidence for the antiepileptic action of NPY in acquired limbic epilepsies has been steadily accumulating from animal studies in vivo (Marksteiner et al., 1989; Erickson et al., 1996; Kofler et al., 1997; Vezzani et al., 1999; Reibel et al., 2001; Noe et al., 2008, 2009; Sorensen et al., 2009) and in vitro (Greber et al., 1994; Marsh et al., 1999), as well as in human studies (Patrylo et al., 1999; Takahashi et al., 1999; Furtinger et al., 2001; Thom et al., 2009). However less work has been done on the effect of NPY in generalized, thalamocortical-based epilepsies, which can respond differently to certain antiepileptic drugs (AEDs) when compared with the limbic epilepsies (Perucca et al., 1998; Stroud et al., 2005; Liu et al., 2006). Our group has shown that NPY injected intracerebroventricularly (i.c.v.) potently suppresses generalized absence-type seizures in the genetic absence epilepsy rats from Strasbourg (GAERS) (Stroud et al., 2005; Morris et al., 2007), with Y2 receptors being most important in mediating this effect (Morris et al., 2007).
Absence seizures are one of the most common types of seizures seen in patients with idiopathic generalized epilepsies, occurring in a number of different syndromes, in particular childhood absence epilepsy (CAE), juvenile absence epilepsy (JAE), and juvenile myoclonic epilepsy (JME) (Commission on Classification and Terminology of the International League Against Epilepsy 1981). Absence seizures are characterized by recurrent nonconvulsive episodes of loss of awareness and responsiveness, commonly accompanied by minor motor manifestations but without loss of postural tone. The electroencephalography (EEG) recording during absence seizures shows bihemispheric, synchronous, generalized spike-and-wave discharges (SWDs) at approximately three cycles per second, which start and end abruptly on an otherwise normal background EEG. GAERS are a well-characterized rat model of idiopathic generalized epilepsy with spontaneous absence-type seizures that are accompanied by generalized SWDs that are morphologically similar to absences seizures in humans, except they have a faster cycle frequency (i.e., 6–8 Hz. compared with 3 Hz) (Danober et al., 1998; Powell et al., 2009). Furthermore, the seizures in GAERS have a pharmacologic response profile similar to those of human absence seizures (Danober et al., 1998). The neurophysiologic basis of absence seizures in GAERS, and in humans, is sustained oscillatory firing within the thalamocortical circuit (Danober et al., 1998). The circuit consists of reciprocally innervated excitatory corticothalamic and thalamocortical glutaminergic neurons in the cortex and ventrobasal thalamus (VB), and γ-aminobutyric acid (GABA)ergic inhibitory neurons in the thalamic reticular nucleus (nRT) and the cortex (Danober et al., 1998). Focal administration of AEDs into different areas of the thalamocortical circuit can suppress or aggravate the seizures in GAERS (Gurbanova et al., 2006; Liu et al., 2006; Gulhan Aker et al., 2006; Polack & Charpier, 2009; Polack et al., 2009).
This study aimed to investigate the site within the thalamocortical circuit that NPY most potently acts to exert its anti–absence seizure effect in GAERS. The effects on spontaneous seizures in freely moving rats were measured of focal microinjections of NPY into key regions of the circuit: the primary somatosensory cortex (S1), the secondary somatosensory cortex (S2), the (VB), or the caudal nRT.
- Top of page
- Materials and Methods
This study investigated the sites within the thalamocortical circuitry in which NPY acts to suppress seizures in the GAERS model of absence epilepsy. The most marked antiseizure effect was seen when NPY was focally injected into the somatosensory cortex, with the amount of seizure suppression following injection into the S2 region significantly different to that following saline injections in the same region. No seizure suppression was seen following injections into the motor cortex or the thalamic regions. These findings indicate that the somatosensory cortex is most likely to be the primary site in the thalamocortical circuit in which NPY acts to suppress seizures in this animal model of absence epilepsy.
The thalamocortical circuit in humans and rodents is responsible for initiating and maintaining absence seizures (Danober et al., 1998). The circuit involves the nRT, the VB, and the cerebral cortex. The neural mechanisms underlying the generation of absence-related SWDs within the thalamocortical circuit have been widely debated for more than 50 years, with opposing views about whether the cortex, the thalamus, or both have primacy (Pinault & O’Brien, 2007). Over the last decade the “cortical focus” theory has become prominent (Meeren et al., 2002; Polack et al., 2007). Using multisite field recordings in awake and freely moving WAG/Rij rats (a phenotypically similar absence-epilepsy model to GAERS), Meeren et al. (2002) reported that seizures started in the perioral somatosensory region (S1) and then secondarily spread to other cortical and thalamic regions. Polack et al. (2007) performed intracellular electrographic recordings into the facial somatosensory cortex (S1) region of GAERS and reported that the neurons in the deep layers (V and VI) were more ictogenic in nature and also exhibited preictal oscillations compared to motor cortex and ventrobasal complex in the thalamus. Previous work by Pinault has shown that layer VI in the S1 cortex plays an important role in driving the propagation of SWDs (Pinault, 2003; Pinault et al., 2006). Recent work from our group studying oscillatory “seizure-like” network discharges in thalamocortical brain slices from GAERS cultured in vitro, demonstrated, using rapid calcium fluorescence imaging, that the discharges were initiated in the deep cortical layers before secondarily spreading to other cortical regions and the interconnected thalamus (Adams et al., 2011).
Neuropharmacologic studies have also supported the importance of the somatosensory cortical region in generating absence seizures in these rat models. Polack et al. (2009) demonstrated that injecting a sodium channel blocker (tetrodotoxin) focally into the S1 somatosensory cortex in GAERS blocked spontaneous seizures, whereas it did not produce the same effect when delivered to the motor cortex or thalamus. Other groups have also shown that focal injection drugs that suppress neuronal firing into the S1 somatosensory cortex suppresses seizures in either GAERS or WAG/Rij rats, including lidocaine (Sitnikova & van Luijtelaar, 2004) and ethosuximide (Richards et al., 2003; Manning et al., 2004; Gurbanova et al., 2006).
The results reported here provide further support for the importance of the somatosensory cortex in initiating these electroclinically “generalized” seizures in the GAERS model. The study provides several further novel observations. First, this is the first study to demonstrate that an endogenous anticonvulsant, namely NPY, acts primarily in the somatosensory cortex and not the thalamus to suppress seizures, although that it is important to note that pharmacologic rather than physiologic doses were used. Second, although the previous electrophysiologic and physiologic studies had concentrated on the S1 region of the somatosensory cortex, and the relationship of this compared to motor cortical and thalamic regions, this study also investigated the effects of focal NPY injections into the S2 cortical region. It is notable that seizure suppression following injection into S2 was found to be greater than following injection into S1, suggesting that this region may actually be more critical to seizure generation.
This study did not investigate the cellular mechanism of action of NPY to suppress seizures in the somatosensory cortex. Our previously published data using i.c.v. injections of specific Y-subtype agonists and antagonists, indicates that Y2 is the Y receptor subtype that has the strongest seizure-suppressing effects in GAERS, but activation of Y5 and Y1 receptors also suppressed seizures (Morris et al., 2007). However, Y2 receptors have been reported to be expressed at relatively low levels in rat somatosensory cortex compared with Y5 receptors (Parker & Herzog, 1999). It is possible that the pattern of expression of Y receptor subtypes is altered in GAERS, and examining this, along with focal injection of selective agonists for Y-receptor subtypes, is the focus of future work to define the cellular mechanisms mediating the effect of focal NPY injections into the S2 cortex.
In contrast to the effects seen with focal cortical administration, we found that focal injections of NPY into the VB paradoxically aggravated the number of seizures occurring in our GAERS rats, with a strong trend to have an increased total time in seizure postinjection (Fig. 2D,E). This is reminiscent of the effect we also found with focal injections of the AED carbamazepine into this structure in GAERS, which we showed was mediated by GABAA receptors (Liu et al., 2006). We proposed that this resulted in hyperpolarization of thalamocortical neurons within the VB, thereby de-inactivating low threshold calcium channels, making the cells more liable to fire in oscillatory bust firing mode and so promoting absence seizures. Consistent with this, others have shown that enhancing GABA activity locally in the VB thalamus by injection of the GABA transaminase inhibitor, γ-vinyl GABA, aggravates seizures in GAERS (Liu et al., 1991), and that microinjections of the GABAA agonist, muscimol, into the VB enhance spontaneous absence-like seizures in epileptic mice (Hosford et al., 1997). NPY actions at postsynaptic Y1 and Y5 receptors located on somata and dendrites of thalamocortical neurons in the VB enhance hyperpolarization of the VB thalamocortical neurons via activation of G-protein–activated inwardly rectifying potassium (GIRK) channels (Sun et al., 2001; El Bahh et al., 2005), which could therefore also explain the effects of focal NPY administration at this site to aggravate absence seizures in GAERS.
We did not demonstrate any effects on seizures of NPY injections into the caudal nRT. We chose to inject the caudal part of the nRT because this region receives direct inputs from the somatosensory cortex and projects to the ventrobasal thalamic relay nuclei that are primarily involved in SWDs (Vergnes et al., 1987; Pinault & Deschênes, 1998; Pinault, 2003). In contrast, the rostral nRT receives inputs primarily from motor and limbic structures, and projects to the anterior, intralaminar, and midline thalamic nuclei (Pinault & Deschênes, 1998; Aker et al., 2006), regions demonstrated on depth recordings not to be primarily involved in the SWDs (Vergnes et al., 1987). However it is likely that the rostral part of the nRT plays a role in modulating the propagation and maintenance of SWDs in genetic rat models, and a recent lesional study in the WAG/Rij rat suggested that this region may actually be more important for this than the caudal nRT (Aker et al., 2006; Meeren et al., 2009). Focal injections of the GABAA receptor antagonist, bicuculline, into either the rostral or caudal nRT has been shown to have effects that were opposite to those on SWDs in GAERS, with the former suppressing and the latter increasing SWD duration (Aker et al., 2006).
The findings of this study and other studies, indicate that genetically determined, phenotypically “generalized” seizures can be generated by relatively restricted regions of cerebral cortex is of clinical as well as scientific importance. This is reflected in the recently proposed Revised Terminology and Concepts for Organization of Seizures and Epilepsies of the International League Against Epilepsy (Berg et al., 2010) which, moving away from the traditional concept of these seizures being “generalized” from their onset, conceptualizes generalized seizures as “originating at some point within, and rapidly engaging, bilaterally distributed networks. Such bilateral networks can include cortical and subcortical structures, but do not necessarily include the entire cortex.” The clinical relevance is that this potentially opens up the prospect that focal drug delivery systems, administering NPY or other seizure suppressing compounds via mechanical, viral, or stem cell technology, could be applied to control seizures in patients with medically refractory genetic generalized epilepsies, not just those with focal epilepsies.