We have examined the topography of the cerebral cortex of the Australian echidna (Tachyglossus aculeatus), using Nissl and myelin staining, immunoreactivity for parvalbumin, calbindin, and nonphosphorylated neurofilament protein (SMI-32 antibody), and histochemistry for acetylcholinesterase (AChE) and NADPH diaphorase. Myelinated fibers terminating in layer IV of the cortex were abundant in the primary sensory cortical areas (areas S1, R, and PV of somatosensory cortex; primary visual cortex) as well as the frontal cortex. Parvalbumin immunoreactivity was particularly intense in the neuropil and somata of somatosensory regions (S1, R, and PV areas) but was poor in motor cortex. Immunoreactivity with the SMI-32 antibody was largely confined to a single sublayer of layer V pyramidal neurons in discrete subregions of the somatosensory, visual, and auditory cortices, as well as a large field in the frontal cortex (Fr1). Surprisingly, SMI-32 neurons were absent from the motor cortex. In AChE preparations, S1, R, V1, and A regions displayed intense reactivity in supragranular layers. Our findings indicate that there is substantial regional differentiation in the expanded frontal cortex of this monotreme. Although we agree with many of the boundaries identified by previous authors in this unusual mammal (Abbie [1940] J. Comp. Neurol. 72:429-467), we present an updated nomenclature for cortical areas that more accurately reflects findings from functional and chemoarchitectural studies.

We have examined the distribution and morphology of neurons immunoreactive for nonphosphorylated neurofilament protein (SMI-32 antibody), calcium-binding proteins (parvalbumin, calbindin, calretinin), and neuropeptide Y as well as neurons reactive for NADPH diaphorase in the cerebral cortex of the Australian short-beaked echidna (Tachyglossus aculeatus). We have also studied synaptic morphology and density in S1 somatosensory cortex and assessed parameters associated with metabolic activity of the cerebral cortex (vessel volume density, mitochondrial volume density, and mitochondrial numerical density) in semi- and ultrathin sections. SMI-32 immunoreactivity was found mostly in layer V pyramidal neurons in selected cortical regions (S1, PV, V1, A). These neurons often showed atypical morphology compared with therian cortex. Neurons immunoreactive for calcium-binding proteins were broadly similar in both morphology and distribution to those seen in therian cortex, although calretinin-immunoreactive neurons were rare. Both Gray type I and Gray type II synapses could be identified in echidna S1 cortex and were similar to those seen in therian cortex. Peak synaptic density was in upper layer IV, followed by layer I, lower layer II, and upper layer III. Most synapses were of type I (72%), although types I and II were encountered with similar frequency in lower layer II and upper layer III. The capillary volume fraction values obtained for the echidna (from 1.18% in V1 to 1.34% in S1 cortex) fall within the values for rodent cortex. Similarly, values for mitochondrial volume fraction in echidna somatosensory cortex (4.68% +/- 1.76%) were comparable to those in eutherian cortex.

We have used Valverde-Golgi and Golgi-Colonnier techniques to analyze cortical neuronal morphology in four regions (frontal cortex, primary motor cortex, primary somatosensory cortex, primary visual cortex) of the isocortex of the echidna (Tachyglossus aculeatus). Eight classes of neurons could be identified – pyramidal, spinous bipolar, aspinous bipolar, spinous bitufted, aspinous bitufted, spinous multipolar, aspinous multipolar and neurogliaform. All except the pyramidal neurons were morphologically similar to neuronal classes seen in eutherian and metatherian isocortex. Pyramidal neurons made up a small proportion of all cortical neurons encountered in our preparations of echidna cortex (34% in visual cortex, 35% in somatosensory cortex, 41% in frontal cortex and 49% in motor cortex) compared to both reported values in eutherian cortex and values we found in rat cortex impregnations prepared in an identical fashion to the echidna material (75% in rat motor and 78% in rat somatosensory cortex). Many pyramidal neurons in the echidna isocortex were atypical (30–42% depending on region) with inverted somata, short or branching apical dendrites and/or few basal dendrites, very different from the usual pyramidal neuron morphology in eutherian cortex. Dendritic spine density on apical and basal dendrites of echidna pyramidal neurons in somatosensory cortex and apical dendrites of motor cortex pyramidal neurons was also lower than that found in the rat. The present findings are consistent with both pyramidal neurons and the many diverse types of non-pyramidal neurons having already emerged as discrete morphological entities very early in mammalian cortical evolution, at the time of divergence of the therian and prototherian lineage.

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We have examined the distribution of immunoreactivity for GAP-43 in the developing and adult brain of a diprotodontid metatherian, the tammar wallaby ( Macropus eugenii). The distribution of GAP-43 immunoreactivity in the neonatal wallaby brain was strikingly heterogeneous, in contrast to that reported for the newborn polyprotodontid opossum. Immunoreactivity for GAP-43 in the developing wallaby brain showed a caudal-to-rostral spatiotemporal gradient, with the brainstem well in advance of the telencephalon throughout the first 100 days of postnatal life. In many regions examined, GAP-43 immunoreactivity passed through the following phases: 1. intense immunoreactivity in developing fiber tracts and occasional somata; 2. diffuse homogeneous immunoreactivity; 3. selective loss of immunoreactivity in particular nuclei or cortical regions. In the isocortex, selective loss of GAP-43 immunoreactivity in the somatosensory and visual cortex (at postnatal day 115) coincided with the maturation of the laminar distribution of terminal thalamocortical axonal fields. Within adult cortical regions, GAP-43 immunoreactivity was highest in layer I of all regions, lower layers (V and VI) of primary somatosensory and visual cortices, layers II/III of motor and cingulate cortex, and layer IV of entorhinal cortex. Our findings suggest that, while patterning of GAP-43 immunoreactivity in the mature brain is similar across meta- and eutheria, there may be early developmental differences in the distribution of GAP-43 immunoreactivity between poly- and diprotodontid metatheria.