Adam L. Smith

An investigation of the role of auditory cortex in sound localization using muscimol-releasing Elvax.

Lesion studies suggest that primary auditory cortex (A1) is required for accurate sound localization by carnivores and primates. In order to elucidate further its role in spatial hearing, we examined the behavioural consequences of reversibly inactivating ferret A1 over long periods, using Elvax implants releasing the GABAA receptor agonist muscimol. Sub‐dural polymer placements were shown to deliver relatively constant levels of muscimol to underlying cortex for >5 months. The measured diffusion of muscimol beneath and around the implant was limited to 1 mm. Cortical silencing was assessed electrophysiologically in both auditory and visual cortices. This exhibited rapid onset and was reversed within a few hours of implant removal. Inactivation of cortical neurons extended to all layers for implants lasting up to 6 weeks and throughout at least layers I–IV for longer placements, whereas thalamic activity in layer IV appeared to be unaffected. Blockade of cortical neurons in the deeper layers was restricted to ≤ 500 µm from the edge of the implant, but was usually more widespread in the superficial layers. In contrast, drug‐free Elvax implants had little discernible effect on the responses of the underlying cortical neurons. Bilateral implants of muscimol–Elvax over A1 produced significant deficits in the localization of brief sounds in horizontal space and particularly a reduced ability to discriminate between anterior and posterior sound sources. The performance of these ferrets gradually improved over the period in which the Elvax was in place and attained that of control animals following its removal. Although similar in nature, these deficits were less pronounced than those caused by cortical lesions and suggest a specific role for A1 in resolving the spatial ambiguities inherent in auditory localization cues.

Manufacture and release characteristics of Elvax polymers containing glutamate receptor antagonists

Implantable sustained-release polymers offer an alternative to osmotic minipumps for the local delivery of drugs to specific brain areas. Here we describe the production of Elvax polymers containing a range of glutamate receptor antagonists and the quantitative characterization of their release properties. Sections of Elvax (200 or 400 microns), prepared by a dimethyl sulphoxide-based method, containing the NMDA antagonist MK-801 or the non-NMDA antagonist CNQX exhibited similar release profiles: an initial 2-week burst followed by a slow decline in release rate over the next 6 weeks. Differences in slice preparation method and thickness or drug concentration and solubility all led to alterations in the level of drug release, but not the overall exponential nature of the release curve. Elvax sections prepared by an aqueous method containing the NMDA antagonists CPP or APV displayed more constant but much lower levels of release than those from the dimethyl sulphoxide-based method. The in vitro release characteristics were compared with in vivo release of MK-801 and the close correspondence observed indicates that the in vitro release data is an accurate predictor of the drug release behaviour of implanted Elvax slices.

Deafferentation induced changes in GAD67 and GluR2 mRNA expression in mouse somatosensory cortex.

Partial vibrissectomy in adult mice induces body map plasticity in SI barrel cortex. To examine if the disturbed balance of cortical activation affects the excitatory and inhibitory neurotransmitter systems, we studied glutamic acid decarboxylase (GAD 67) and AMPA receptor subunit GluR2 mRNA expression in the barrel cortex. At varying times post-vibrissectomy, sparing row C of whiskers on one side of the snout, the brains were processed for in situ hybridization using specific [(35)S]oligonucleotides to detect the laminar localization of GAD67 and GluR2 mRNAs. Three and seven days after vibrissectomy, the expression of GAD67 was decreased in the deafferented cortex, while 30 days post-lesion, no effects were observed. At 3 days post-lesion, an ipsilateral decrease in GAD67 mRNA expression was also observed. No decreases in GluR2 transcripts were found in the deafferented cortex, but an increased expression was observed in the representation of the spared row C of whiskers 3 days after vibrissectomy. Seven and 30 days post lesion no changes in GluR2 expression were found. These data indicate that in the barrel cortex, peripheral deafferentation transiently regulates GAD67 and GluR2 expression at the transcriptional level. We suggest that this may be a manifestation of adaptive processes.

The development of topographically-aligned maps of visual and auditory space in the superior colliculus.

The role of the superior colliculus in attending and orienting to sensory stimuli is facilitated by the presence within this midbrain nucleus of superimposed maps of different sensory modalities. We have studied the steps involved in the development of topographically-aligned maps of visual and auditory space in the ferret superior colliculus. Injections of fluorescent beads into the superficial layers showed that the projection from the contralateral retina displays topographic order on the day of birth (PO). Recordings made from these layers at the time of eye opening, approximately 1 month later, revealed the presence of an adult-like map of visual space. In contrast, the auditory space map in the deeper layers emerged gradually over a much longer period of postnatal life. In adult ferrets in which one eye had been deviated laterally just before eye opening, the auditory spatial tuning of single units recorded in the contralateral superior colliculus was shifted by a corresponding amount, so that the registration of the visual and auditory maps was maintained. Chronic application of the NMDA-receptor antagonist MK801 disrupted the normal development of the auditory space map, but had no effect on the visual map in either juvenile or adult animals, or on the auditory map once it had matured. These findings indicate that visual cues may play an instructive role, possibly via a Hebbian mechanism of synaptic plasticity, in the development of appropriately tuned auditory responses, thereby ensuring that the neural representations of both modalities share the same coordinates. Changes observed in the auditory representation following partial lesions of the superficial layers at PO suggest that these layers may provide the source of the visual signals responsible for experience-induced plasticity in auditory spatial tuning.

SPATIOTEMPORAL PATTERNING OF GLUTAMATE RECEPTORS IN DEVELOPING FERRET STRIATE CORTEX

We have studied glutamate receptor levels during very early phases of cortical formation by using quantitative in vitro autoradiography to map the expression of NMDA, AMPA and kainate receptors in the developing primary visual cortex of the ferret. NMDA and non‐NMDA receptors exhibit very different developmental profiles in primary visual cortex. NMDA receptor density is low at birth and increases throughout the first 2 postnatal months, rising between threefold (layers II/III) and ninefold (layer VI). In contrast, AMPA receptors are abundant at birth and their density remains constant for the first postnatal month, before rising by a maximum of 1.7‐fold (layer I) at around the time of eye‐opening (postnatal day 32). Kainate receptors are also present in high levels at birth and their expression levels rise in the early postnatal period by between 1.5‐fold (layer I) and threefold (layers V/VI) to a peak just after eye‐opening. The proportion of the total ionotropic glutamate receptor binding contributed by NMDA receptors thus rises from 5% at birth to a maximum of 22% at 2 months of age, while the AMPA receptor contribution falls from 87% to 72% over the same period. Below cortex, all three glutamate receptor subtypes are expressed in the subplate region for the first 3 postnatal weeks. These developmental patterns, combined with the fact that AMPA receptors are densely expressed in the proliferative zones underlying presumptive area 17, indicate that non‐NMDA receptor expression levels in primary visual cortex are mostly specified much earlier than those of NMDA receptors.

Development of laminar distributions of kainate receptors in the somatosensory cortex of mice

Kainate receptors were present at birth in the murine somatosensory cortex as revealed by quantitative in vitro autoradiography. During the first five postnatal days [3H]kainate binding rapidly increased and the maximum density in layer IV was reached at P12. The adult laminar pattern of receptor binding distribution was established by the third postnatal week with the heaviest labeling of infragranular layers. The sharp increase of kainate receptor during the first postnatal week coincides with the critical period for cytoarchitectonic plasticity of the barrels and establishment of functional thalamo-cortical connections in the barrel field.

Genetically induced retinal degeneration leads to changes in metabotropic glutamate receptor expression

In the retina, neurotransmission from photoreceptors to ON-cone and rod bipolar cells is sign reversing and mediated by the metabotropic glutamate receptor mGluR6, which converts the light-evoked hyperpolarization of the photoreceptors into depolarization of ON bipolar cells. The Royal College of Surgeons (RCS) rat retina undergoes progressive photoreceptor loss due to a genetic defect in the pigment epithelium cells. The consequences of photoreceptor loss and the concomitant loss of glutamatergic input to second-order retinal neurons on the expression of the metabotropic glutamate receptor was investigated in the RCS rat retina from early stages of photoreceptor degeneration (P17) up to several months after complete rod and cone degeneration (P120). The expression of the gene encoding mGluR6 was studied by in situ hybridization in the retina, using an [(35)S]dATP-labeled oligonucleotide probe. In congenic control and RCS retina, we found mRNA expression of mGluR6 receptor only in the outer half of the inner nuclear layer (INL) on emulsion-coated retinal sections. Quantitative analysis of the hybridization signal obtained from the autoradiographic films revealed decreased expression levels of the mGluR6 mRNA at early stages of photoreceptor degeneration (P17). On the contrary, increased expression levels were observed at late stages of degeneration (P60 and P120) in RCS compared to congenic control retina. In conclusion, our data demonstrate that the metabotropic glutamate receptor-6 mRNA levels are altered in the young and adult RCS rat retina and suggest that the genetically induced degeneration of photoreceptors affects the expression of this receptor by the INL retinal neurons.