I think this needs to be elaborated a bit more. Even after this change you can still have names like _, _..._, a.....a, etc.

Maybe we want something like the following:

(?!\.)[a-zA-Z0-9]*([._]?[a-zA-Z0-9]+)*(?<!\.)

This enforces minimum one of a-zA-Z0-9 and allows one of the special chars [._] in a row.

I'm not sure but with enforcing one character behind the special chars, the negative lookbehind could also be removed.
At the moment underscores aren't possible too at the end.

Another Option:

(?!\.)[a-zA-Z0-9]*([._]?[a-zA-Z0-9]+[._]?)*(?<!\.)

Hmm, should be alphanumeric and _ and ., where those 2 characters can't be by themselves or the beginning nor end of the username

I don't understand what you mean by "where those 2 characters can't be by themselves".
Do you mean, that we don't to allow the examples of @Komposten, but we allow something link a.b.c_d_e?

Since we're checking a bounded string there is no reason to have lookaheads and lookbehinds. Better to define a concrete regex for the pattern we want, rather than trying to match what we don't want.

To that end we can simply the regex to something like this:

^[a-zA-Z0-9](?:[._]?[a-z-A-Z0-9])*$

Basically, always start with at least one alphanumeric character.
Follow that up with zero or more alphanumeric characters, each of which may be preceded by exactly one period or underscore.

I haven't done extensive testing, but here are some examples (highlight = match):

@Komposten thanks for the small fixes to my proposed regex. :)
@lifenautjoe how will we handle usernames that exist that aren't allowed with the new check anymore? I also found a username with a é, which isn't allowed.